1,721,029 research outputs found
Proteomic Analysis of Nuclear HBV rcDNA Associated Proteins Identifies UV-DDB as a Host Factor Involved in cccDNA Formation
Get PDFIndiana University-Purdue University Indianapolis (IUPUI)Despite the lifecycle of the hepatitis B virus (HBV) being extensively investigated and described, there remains a significant gap in our knowledge of arguably one of the most crucial steps in the HBV lifecycle, the formation and maintenance of a covalently closed circular DNA (cccDNA) reservoir. Advancements in our understanding of host factors and pathways involved in cccDNA formation have been made through hypothesis driven studies and shRNA/siRNA screenings. We sought to create a targeted-unbiased assay to directly observe host factor-rcDNA interactions. This was achieved through an rcDNA Co-Immunoprecipitation paired Mass Spectrometry (rcDNA-CoIP/MS) assay. We created a DNA oligo complimentary to the open portion of the HBV rcDNA, labeled with biotin, to facilitate easy precipitation of nuclear rcDNA and complexed proteins. Proteins precipitated were analyzed through liquid chromatography paired mass spectrometry (LC/MS). Along with previously reported host factors, several factors of DNA damage repair pathways/complexes were also identified. A component of the UV-DDB complex, DDB1, surfaced as a hit. UV-DDB/rcDNA binding was confirmed through ChIP-qPCR. DDB2, the DNA damage binding component of the UV-DDB complex was knocked out in HepG2-NTCP and HepAD38 cells. This resulted in a significant decrease in the formation of cccDNA in DDB2 knockout cell lines following infection or induction. The subsequent reduction of downstream indicators of cccDNA formation such as viral RNA and proteins, HBcAg and HBeAg, showed a consistent decrease with cccDNA levels. Ectopic expression of DDB2 in the knockout cell lines rescued HBV phenotypes of cccDNA levels and its downstream indicators. Inactive mutant DDB2 plasmids were also transfected into the DDB2 K/O cell lines and failed to rescue cccDNA indicators. We therefore showed through a novel assay that we can discover novel viral rcDNA-host interactions, such as the UV-DDB complex recruiting DNA repair pathways to “repair” rcDNA to cccDNA
Conserved Residues in Murine Papillomavirus E2 Regulate the Viral Life Cycle
No full textIUIPapillomaviruses (PVs) are small, non-enveloped DNA viruses that infect the
stratified epithelia. Once an infection is initiated, the virus must successfully navigate the
three stages of its life cycle: establishment, maintenance, and vegetative amplification. A
major mechanism of regulating this viral program is post-translational modification on
the viral E2 protein, which is responsible for orchestrating viral transcription, replication,
and genome partitioning.
The hypothesis underscoring this work is that residues in E2 are highly conserved
across PV types because they serve some structural or functional purpose for the virus. A
targeted mutant library was generated in E2 from murine papillomavirus (MmuPV1) to
investigate conserved residues that have been shown to be post-translationally modified
in the E2 of other PVs, including BPV-1 and high-risk HPV-31. In the transactivation
domain (TAD) tyrosine 102 and the lysine 112/113 motif were modified to their
constitutively modified (phosphorylated and acetylated, respectively) or unmodified
states, while cysteine 307 in the DNA binding and dimerization domain (DBD) was
mutated to a less-reactive serine or DNA binding defective phenylalanine mutant. We
characterized how mutation at each of these conserved sites alters E2 function using a
battery of in vitro assays to assess for transcription and replication ability. We also
studied how each mutant contributes to disease progression using an
immunocompromised mouse model assessing cutaneous disease. We demonstrate that mutants which fail to replicate transiently in vitro will also fail to induce proliferative
wart formation, establishing a predictive link between in vitro and in vivo experiments.
Taken together, our findings suggest that modifications on conserved residues in E2 act
as molecular switches that regulate E2 activity throughout the cellular and viral life cycle
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Regulation of papillomavirus E2 protein by posttranslational modification
Get PDFIndiana University-Purdue University Indianapolis (IUPUI)Papillomaviruses (PVs) are small, double-stranded DNA viruses. Hundreds of species have evolved to replicate in mammals, birds, and reptiles. Approximately two hundred species are estimated to infect humans alone, and these human papillomaviruses (HPVs) cause diseases ranging from benign warts to anogenital and oropharyngeal cancers. While vaccination is effective at preventing the majority of these infections and their disease outcomes, there are no successful treatments for existing infections; thus, exploration of novel therapeutic targets is warranted.
PVs control expression and function of their gene products through alternative splicing, alternate start codons, and post-translational modification (PTM). The viral E2 protein regulates transcription, replication, and genome maintenance in infected cells, and PTMs have been demonstrated for E2 proteins from multiple papillomavirus types. Serine phosphorylation events were reported to influence E2 stability, and our laboratory was the first to describe in vitro acetylation events with implications for E2 transcription function. Here we report confirmation of these acetylation events in vivo and additional data elucidating the role of these PTMs in viral transcription. Moreover, we present a novel phosphorylation site for bovine papillomavirus type 1 (BPV-1) E2 at tyrosine 102 (Y102). Using phospho-deficient and phospho-mimetic point mutants, we found that this site influences E2-mediated transcription and replication, and we hypothesize that phosphorylation at Y102 regulates these activities by interrupting the association of E2 with its binding partners. We also report interaction of BPV-1 E2 and HPV-31 E2 with different receptor tyrosine kinases (TKs), most notably members of the fibroblast growth factor receptor family. We hypothesize that Y102 phosphorylation by these receptors occurs early in infection to limit viral replication and gene expression. Further studies will cement the role of RTKs in PV biology and could reveal novel therapeutic strategies
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Biological Functions of Intracellular Hepatitis B e Antigen
Get PDFIndiana University-Purdue University Indianapolis (IUPUI)The function(s) of the intracellular form of HBeAg, previously reported as the
preCore protein intermediate (p22) without the N-terminal signal peptide, remains elusive.
Here, we propose to elucidate the translocation of p22 during its formation from
endoplasmic reticulum (ER) to cytosol, how it differs from core in its inability to form a
capsid and the biological functions of cytoplasmic and nuclear p22. Firstly, we have
identified that a portion of p22, after the cleavage of its signal peptide in ER, is released
back into the cytosol through an ERAD-independent mechanism, as neither wildtype nor
dominant-negative p97 affected the ER-to-cytosol translocation of p22 or ER-Golgi
secretion of HBeAg. Secondly, despite sharing the same sequence with core protein except
for the extended 10 amino acid precore region at the N-terminus, we observed that p22
wildtype and C-7Q mutant are unable to form a capsid. Thirdly, we report that p22 but not
the secreted HBeAg significantly reduced interferon stimulated response element (ISRE)
activity and expression of interferon stimulated genes (ISGs) upon interferon-alpha (IFN-
α) stimulation. Furthermore, in line with this, RNA-seq analysis of ISG induction profile
from IFN-α treated patients showed that HBeAg(+) patients exhibited reduced and weak
antiviral ISG upregulations compared to HBeAg(-) patients. Further, mechanistic study
indicated that while p22 did not alter the total STAT1 or p-STAT1 levels in IFN-α treated
cells, it blocked the nuclear translocation of p-STAT1 by interacting with karyopherin α1,
indicating that the cytoplasmic p22 may impede JAK-STAT signaling to help the virus
evade host innate immune response and cause resistance to IFN therapy in patients.
Additionally, nuclear p22 and nuclear core were found to interact with the promoter regions (ISRE – containing) of ISGs, suggesting a new mechanism of inhibition of ISG expression
upon stimulation. Finally, we found that the nuclear p22 can bind to cccDNA
minichromosome and affects cccDNA maintenance and/or transcription. Thus, our results
indicate that there is a novel ER sorting mechanism for the distribution of the intracellular
and secretory HBeAg, and the intracellular HBeAg may contribute to HBV persistence by
interfering with IFN-α elicited JAK-STAT signaling and regulating cccDNA metabolism
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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