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Zika virus-induced lysis of cervical cancer cells
Title
Zika virus-induced lysis of cervical cancer cells.
Creator
Krishnapura, Harini, Alexander, Kenneth, Parks, Griffith, Jewett, Mollie, University of Central Florida
Abstract / Description
Cervical cancer is the fourth most frequent cancer in women with an estimated 570,000 new cases globally in 2018. Treatment of advanced cervical cancer is often unsuccessful leading to high cancer-related mortality rates, especially in under-resourced countries. Recently, a possible role for the cell surface glycoprotein CD24 in host cell specificity of Zika virus was reported. As an extension of this work, Zika viruses have been proposed as oncolytic therapy for the treatment of...
Show moreCervical cancer is the fourth most frequent cancer in women with an estimated 570,000 new cases globally in 2018. Treatment of advanced cervical cancer is often unsuccessful leading to high cancer-related mortality rates, especially in under-resourced countries. Recently, a possible role for the cell surface glycoprotein CD24 in host cell specificity of Zika virus was reported. As an extension of this work, Zika viruses have been proposed as oncolytic therapy for the treatment of neuroblastoma and other CD24 positive tumors. To determine the permissiveness of cervical cancer cells to Zika virus infection and its association with CD24, we assessed cytopathic effect (CPE) induced by Zika virus in cervical cancer cell lines (HeLa, SiHa and CaSki) by light microscopy and by cytotoxicity assay. Cervical cancer cells were susceptible to Zika virus-induced apoptosis. Upon infection, the morphology of cervical cancer cells changed, exhibiting Zika virus-induced CPE. Cervical cancer cell expression of viral non-structural protein 1 (NS1) after infection demonstrated viral protein translation. Quantitative plaque assays demonstrated the production of competent virions. Because CD24 expression was found to be important for Zika virus infection in neuroblastoma cells, CD24 expression was assessed in cervical cancer cells. Cervical cancer cells expressed low but measurable levels of CD24 mRNA and protein. siRNA-mediated knockdown of CD24 resulted in reduced NS1 expression and reduced levels of virus-induced apoptosis. Taken together our data suggest a possible role for CD24 in Zika virus-induced apoptosis in cervical cancer cells. Zika virus-induced apoptosis of cultured cervical cancer cells presents the possibility for the use of Zika virus as a potential oncolytic therapy for cervical cancer.
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Date Issued
2019
Identifier
CFE0007480, ucf:52682
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007480
ATP-Induced Disassembly of CDTB/CDTC Heterodimer of Cytolethal Distending Toxin
Title
ATP-Induced Disassembly of CDTB/CDTC Heterodimer of Cytolethal Distending Toxin.
Creator
Huhn, George, Teter, Kenneth, Cole, Alexander, Jewett, Mollie, University of Central Florida
Abstract / Description
Cytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi. This fastidious pathogen is the causative agent of genital cancroid. CDT is a heterotrimeric toxin with an AB2 structure consisting of a cell-binding (")B(") domain (CdtA + CdtC) and a catalytic (")A(") domain (CdtB) that has DNase activity. This toxin assembles in the bacterial periplasm that lacks ATP and is secreted into the extracellular environment. After cell...
Show moreCytolethal distending toxin (CDT) is a virulence factor produced by many Gram-negative bacteria, including Haemophilus ducreyi. This fastidious pathogen is the causative agent of genital cancroid. CDT is a heterotrimeric toxin with an AB2 structure consisting of a cell-binding (")B(") domain (CdtA + CdtC) and a catalytic (")A(") domain (CdtB) that has DNase activity. This toxin assembles in the bacterial periplasm that lacks ATP and is secreted into the extracellular environment. After cell binding, CDT is internalized by endocytosis and travels through the endosomes and Golgi before arriving in the endoplasmic reticulum (ER). CdtA is lost from the holotoxin before reaching the Golgi, and CdtB separates from CdtC in the ER. CdtB is then transported into the nucleus, inducing cell cycle arrest and apoptosis. Using disassembly of the AB5 pertussis toxin as a model, we explore that ATP, which is present in the ER lumen but not in the endosomes or Golgi, will cause dissociation of the CdtB/CdtC heterodimer. We have cloned and purified the three individual subunits of the H. ducreyi CDT. When combined, the subunits form a lethal holotoxin. Examining the individual toxin subunits, only CdtB binds with ATP but does not function as an ATPase. CdtB's binding to ATP also does not cause global changes to its secondary structure. After isolating the CdtB/CdtC heterodimer, we have shown the addition of ATP causes CdtC to dissociate from CdtB. The work presented in this Thesis provides a molecular basis for why the CdtB/CdtC heterodimer disassembles after reaching the ER and confirms the novel two-stage disassembly mechanism for CDT, a first in the AB toxin field.
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Date Issued
2019
Identifier
CFE0007657, ucf:52488
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007657
It takes two to tango: the toxin-chaperone relationship
Title
It takes two to tango: the toxin-chaperone relationship.
Creator
Kellner, Alisha, Teter, Kenneth, Moore, Sean, Cole, Alexander, Harper, James, University of Central Florida
Abstract / Description
Cholera toxin (CT) enters the cell via receptor-mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit (CTA1) is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP-ribosylation of its G-protein target. We have shown that the cytosolic chaperones Hsp90 and Hsc70 are required for CTA1 translocation to the cytosol. We have also shown that both are...
Show moreCholera toxin (CT) enters the cell via receptor-mediated endocytosis and travels in a retrograde fashion to the endoplasmic reticulum (ER). The catalytic A1 subunit (CTA1) is then displaced from the rest of the holotoxin, unfolds, and is exported to the cytosol where it regains an active conformation for the ADP-ribosylation of its G-protein target. We have shown that the cytosolic chaperones Hsp90 and Hsc70 are required for CTA1 translocation to the cytosol. We have also shown that both are able to independently bind and refold CTA1. Using libraries of CTA1-derived peptides, we have identified a single Hsc70 binding site, YYIYVI (CTA1 83-88), within the 192 amino acid protein, as well as two distinct Hsp90 binding sites: an N-terminal RPPDEI (CTA111-16) motif and a C-terminal LDIAPA (CTA1 153-158) motif. The LDIAPA motif is unique to CTA1, but an RPPDEI-like motif is present in four other ER-translocating ADP-ribosylating toxins: pertussis toxin, Pseudomonas aeruginosa exotoxin A, Escherichia coli heat-labile toxin, and Salmonella typhimurium ADP-ribosylating toxin. Using site-directed mutagenesis to further investigate the RPPDEI motif, we found that a modification of either proline residue blocks CTA1 translocation to the cytosol. Our work has identified, for the first time, specific amino acid sequences that are recognized by Hsp90/Hsc70 and are essential for toxin translocation from the ER to the cytosol. CT does not require prolyl isomerases for cellular activity, as is the case for Hsp90-dependent endosome-translocating toxins. We therefore hypothesize that the one or both of the prolines within the RPPDEI motif of CTA1 undergo an isomerization event as CTA1 unfolds in the ER. Furthermore, we predict that the trans- to cis- conformational change of proline(s) is the molecular determinate for the atypical Hsp90 interaction observed with CTA1 and related toxins. Additionally, we have identified Hsp90 and other host factors required for the translocation of pertussis toxin.
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Date Issued
2019
Identifier
CFE0007661, ucf:52500
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0007661
Antimicrobial Peptide Resistance and Immunomodulation by HIV-1 gp41
Title
Antimicrobial Peptide Resistance and Immunomodulation by HIV-1 gp41.
Creator
Wood, Matthew, Cole, Alexander, Chai, Karl, Teter, Kenneth, Parkinson, Christopher, University of Central Florida
Abstract / Description
Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide (ENF), are peptides designed to mimic, and thereby competitively inhibit, the viral fusion protein gp41. An exception to this is a class of cyclic, cationic, antimicrobial peptides known as ?-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity....
Show moreFusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide (ENF), are peptides designed to mimic, and thereby competitively inhibit, the viral fusion protein gp41. An exception to this is a class of cyclic, cationic, antimicrobial peptides known as ?-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the ?-defensin analog RC-101 is being developed as a microbicide to prevent sexual transmission of HIV-1. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development.Partial drug resistance due to genetic variability within HIV-1 presents a major hurdle in microbicide development. Drug-resistance mutations, whether naturally occurring or resulting from selection during treatment, often apply to many drugs in a particular class. Combining different drug classes into a single microbicide should provide greater protection against the growing variability observed in HIV. Our work has identified the beneficial effects of combining the fusion inhibitor RC-101 and the RT inhibitor CSIC to prevent transmission of clinically isolated and drug-resistant HIV-1.Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. While this chemotactic activity has been characterized, it is not known how these peptides could be produced under biological conditions. Our findings demonstrate that the epithelial serine protease matriptase efficiently cleaves the gp41 HR1 region at conserved residues into a chemotactic peptide.Here, we present evidence that advances our understanding of resistance to peptide entry inhibitors, reveals a potential benefit to combining specific drugs in an antiviral microbicide, and identifies a pathway by which HIV-1 may generate peptides to exploit host immunity. This work thereby facilitates improved methods in countering drug resistance and the development of new antiviral approaches to prevent HIV-1 transmission. Additionally, we have revealed basic mechanistic evidence that shed light on our current understanding of HIV-1 infection. Specifically, our focus on gp41 provides much needed insight into its role in membrane fusion, drug susceptibility, and modification of host responses.
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Date Issued
2014
Identifier
CFE0005560, ucf:50286
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005560
The role of a highly conserved eubacterial ribosomal protein in translation quality control
Title
The role of a highly conserved eubacterial ribosomal protein in translation quality control.
Creator
Naganathan, Anusha, Moore, Sean, Cole, Alexander, Teter, Kenneth, Roy, Herve, Koculi, Eda, University of Central Florida
Abstract / Description
The process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of...
Show moreThe process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of decoding, making it difficult to envision how it might affect decoding and reading frame maintenance. Twenty years after the identification of L9's putative function, there is no mechanism for how a remotely located L9 improves translation fidelity. This mystery makes our picture of translation incomplete. Despite the high conservation of L9 in eubacteria, E.coli lacking L9 does not exhibit any obvious growth defects. Thus, the evolutionary advantage conferred by L9 in bacteria is masked under laboratory conditions. In order to uncover unique L9-dependent conditions, a library of E. coli mutants was screened to isolate those that rely on L9 for fitness. Interestingly, factors found to be synergistic with L9 had no known role in fidelity. Six independent mutants were isolated, each exhibiting a severe growth defect that is partially suppressed in the presence of L9. One class of L9-dependent mutations was present in an essential ribosome biogenesis factor, Der. Der's established function is in the maturation of the large ribosomal subunit. The identified mutations severely impaired the GTPase activity of Der. Interestingly, L9 did not directly compensate for the defective GTPase activity of mutant Der. The second class of L9-dependent mutations was present in EpmA and EpmB, factors required to post-translationally modify elongation factor, EF-P. EF-P's established function is in the translation of poly-proline containing proteins. EF-P deficient cells were nearly inviable in the absence of L9; however, L9 did not directly influence poly-proline translation. Therefore, in each case, L9 improved cell health without altering the activity of either Der or EF-P. Remarkably, the der mutants required only the N domain of L9, whereas the absence of active EF-P required full-length, wild-type L9 for growth complementation. Thus, each mutant class needed a different aspect of L9's unique architecture. In cells lacking either active EF-P or Der, there was a severe deficiency of 70S ribosomes and the indication of small subunit maturation defects, both of which worsened upon L9 depletion. These results strongly suggest that L9 plays a role in improving ribosome quality and abundance under certain conditions.Overall, the genetic screen lead to the discovery that bacteria need L9 when either of two important translation factors (Der or EF-P) is inactivated. This work has characterized the physiological requirement for L9 in each case and offers a new insight into L9's assigned role in translation fidelity.
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Date Issued
2015
Identifier
CFE0005674, ucf:50169
Format
Document (PDF)
PURL
http://purl.flvc.org/ucf/fd/CFE0005674