Host cell restriction factors that limit transcription and replication of human papillomavirus

The life cycle of human papillomaviruses (HPV) is tightly regulated by the differentiation state of mucosal and cutaneous keratinocytes. To counteract viral infection, constitutively expressed cellular factors, which are defined herein as restriction factors, directly mitigate viral gene expression and replication. In turn, some HPV gene products target these restriction factors and abrogate their anti-viral effects to establish efficient gene expression and replication programs. Ironically, in certain circumstances, this delicate counterbalance between viral gene products and restriction factors facilitates persistent infection by HPVs. This review serves to recapitulate the current knowledge of nuclear restriction factors that directly affect the HPV infectious cycle

Differential regulation of innate immune cytokine production through pharmacological activation of Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) in burn patient immune cells and monocytes

Burn patients suffer from immunological dysfunction for which there are currently no successful interventions. Similar to previous observations, we find that burn shock patients (≥15% Total Burn Surface Area (TBSA) injury) have elevated levels of the innate immune cytokines Interleukin-6 (IL-6) and Monocyte Chemoattractant Protein-1 (MCP-1)/CC-motif Chemokine Ligand 2(CCL2) early after hospital admission (0–48 Hours Post-hospital Admission (HPA). Functional immune assays with patient Peripheral Blood Mononuclear Cells (PBMCs) revealed that burn shock patients (≥15% TBSA) produced elevated levels of MCP-1/CCL2 after innate immune stimulation ex vivo relative to mild burn patients. Interestingly, treatment of patient PBMCs with the Nuclear Factor-Erythroid-2-Related Factor 2 (NRF2) agonist, CDDO-Me(bardoxolone methyl), reduced MCP-1 production but not IL-6 or Interleukin-10 (IL-10) secretion. In enriched monocytes from healthy donors, CDDO-Me(bardoxolone methyl) also reduced LPS-induced MCP1/CCL2 production but did not alter IL-6 or IL-10 secretion. Similar immunomodulatory effects were observed with Compound 7, which activates the NRF2 pathway through a different and non-covalent Mechanism Of Action (MOA). Hence, our findings with CDDO-Me(bardoxolone methyl) and Compound 7 are likely to reflect a generalizable aspect of NRF2 activation. These observed effects were not specific to LPS-induced immune responses, as NRF2 activation also reduced MCP-1/CCL2 production after stimulation with IL-6. Pharmacological NRF2 activation reduced Mcp-1/Ccl2 transcript accumulation without inhibiting either Il-6 or Il-10 transcript levels. Hence, we describe a novel aspect of NRF2 activation that may contribute to the beneficial effects of NRF2 agonists during disease. Our work also demonstrates that the NRF2 pathway is retained and can be modulated to regulate important immunomodulatory functions in burn patient immune cells

Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo

Meeting Abstracts: Proceedings of the Thirteenth International Society of Sports Nutrition (ISSN) Conference and Expo Clearwater Beach, FL, USA. 9-11 June 201

Sp100 is found inside viral replication factories in close association with viral DNA.

<p>1A cells cultured on glass coverslips were transfected with HPV16 E1 and E2 expression vector (400 ng each) and 50 ng recircularized HPV16 genome. Cells were induced 24 hours after DNA transfection with 3 μM CdSO₄ for four hours and fixed with 4% PFA for confocal microscopy analysis. A. Combined FISH-IF for HPV16 DNA (green), Sp100 protein (red) and EE-E1 (blue). Nuclei (outlined in light blue) were detected with DAPI counterstain. 35 cells were imaged and figure is representative of a single FISH-IF experiment. Images were obtained from a single optical slice. B. Combined FISH-IF for HPV16 DNA (green) and Sp100 protein (red). Nuclei (outlined in light blue) were detected with DAPI counterstain. Magnification of boxed area demonstrates Sp100 (red) association with viral DNA (green). Images shown are single, deconvolved slices obtained from z-stacks collected throughout the nucleus in 0.13 μm per slice. C. 3D reconstruction of panel B showing viral DNA and Sp100 in proximity to each other inside the replication factory. Surface-rendering was generated in IMARIS from a z-stack image (0.13 μm slices) collected at optimum X, Y and Z settings and deconvolved. Images for B and C are representative of three independent experiments with a minimum of 20 cells per experiment analyzed.</p

Sp100 represses late HPV mRNA transcription.

<p><b>A.</b> Timeline of experiment. CIN612-9E cells were transfected with 20 nM siRNA to Sp100 and grown as shown. RNA was isolated at T = 0, 2, 4 and 6 days. <b>B.</b> Cellular transcripts for Sp100, involucrin and filaggrin were measured at the time points indicated. <b>C.</b> Viral transcripts were measured using qPCR with primers that overlapped the splice sites of E6*I, E2 877^2646, E1^E4 and L1. All results were obtained from three independent experiments, each of which contained two technical replicates. <b>Error bars represent +/- SEM. *, P<0.05; **, P<0.005; ***, P<0.0005; ****, P<0.00005; ns, not statistically significant.</b></p

Viral DNA amplification initiates in cells containing Sp100 foci.

<p>Normal cervix and CIN-I HPV16-infected cervical tissue sections were subjected to antigen-retrieval followed by combined FISH-IF (Red: Sp100, Cyan: HPV16 DNA, Blue: DAPI, Green, E4) for confocal microscopy analysis. A. Images showing full thickness sections of stained cervical epithelia. Arrows indicate examples of cells containing both amplifying HPV genomes and Sp100 foci. B. Left, 2D view of a single focal plane of a keratinocyte at the onset of viral DNA amplification with pronounced Sp100 colocalization. Center, 3D view of Z-stack from the same image. Right, 3D reconstruction from the Z stack with rendered surfaces for nucleus (Gray, DAPI), Sp100 (Red), and HPV16 DNA (Cyan) showing Sp100 foci adjacent and surrounded by HPV16 DNA. C. Quantitation of experiment shown in B. 3D images of cells containing both Sp100 foci and HPV DNA were visually assessed and the association of Sp100 foci and HPV DNA signal categorized as not associated, adjacent, or engulfed (Sp100 foci surrounded by HPV DNA signal). Results are from two independent experiments with a total of N = 76 cells counted.</p

PML and Sp100 association with HPV16 E1/E2 replication foci increases in the presence of replicating viral DNA.

<p><b>A.</b> HFK 1A cells cultured on glass coverslips were transfected with HPV16 E1 and E2 expression vectors (400 ng each) and 50 ng of pKS (empty vector), pKS-16Ori, or recircularized HPV16 genome. E1 and E2 expression was induced 24 hours post-transfection with 3 μM CdSO₄ for four hours and cells were fixed with 4% PFA for analysis by indirect immunofluorescence. Cells were stained with EE (E1, green), PML (red) or Sp100 (cyan) antibodies. Nuclei (outlined in light blue) were detected with DAPI counterstain. Images are single optical slices collected by confocal microscopy, and are representative of three independent transfections. B. E1-foci positive cells were scored for association with PML by visually assessing PML’s proximity to the E1-foci. Results were calculated from a minimum of 20 cells per experiment derived from three independent experiments (Total N = 60). Error bars represent +/-SEM.</p

Sp100 represses differentiation dependent genome amplification.

<p><b>A.</b> Southern blot analysis of 9E cell DNA extracted from cells treated with either control, PML or Sp100 siRNA for two days before addition of 1.5 mM CaCl₂ to induce differentiation. DNA was collected at 0 or 96 hours post addition of CaCl2. Prior to electrophoresis, DNA was digested with a restriction enzyme that left supercoiled viral DNA intact (-), or with HindIII to linearize the viral genome (+). DNA was separated by agarose gel electrophoresis and probed with ³²P-labeled HPV 31 genome. The blot shown is representative of four total Southern blots. <b>B.</b> DNA qPCR was performed on CIN612-9E cells treated according to the scheme shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006660#ppat.1006660.g006" target="_blank">Fig 6A</a>. Results shown were averaged from three independent experiments, each of which contained two technical replicates. <b>Error bars represent +/- SEM. *, P<0.05.</b></p

Sp100 binding to HPV chromatin correlates with differentiation-dependent viral functions.

<p>Chromatin immunoprecipitation (ChIP) was performed with samples from growing and differentiated CIN612-9E cells using rabbit IgG and Sp100 antibody. Viral DNA was quantified with qPCR using primers targeting major regions of the HPV31 genome. <b>A.</b> Linear representation of HPV31 genome displaying primer positions (orange boxes) for the upstream regulatory region, early promoter, early region, and late region. <b>B.</b> ChIP binding signals were expressed as a percentage of Sp100 coimmunoprecipitated viral DNA (minus that precipitated with a background IgG antibody) DNA relative to total viral DNA in ChIP input (% Input). Binding signals were averaged from three independent experiments, and the amounts shown are from the equivalent of 2 μg input chromatin. Error bars represent +/- SEM. Statistical significance was calculated using a paired Student t-tests. SD. **, P<0.005; ****, P<0.00005. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006660#ppat.1006660.s003" target="_blank">S3 Fig</a> shows Sp100 protein levels, and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006660#ppat.1006660.s004" target="_blank">S4 Fig</a> shows background IgG binding levels, and histone H3 levels.</p

SAND domain containing isoforms of Sp100 repress viral transcription and replication.

<p><b>A.</b> qPCR was performed on cDNA prepared from HFKs coelectroporated with 2μg of recircularized HPV18 genome and 1μg of an empty expression vector or a vector expressing either Sp100A, B, C, HMG or the Sp100B-DBD mt. Samples were harvested 72 hours post DNA transfection. The spliced viral messages E1^E4 (solid bars) or E6*I (hashed bars) were detected using specific primers for the spliced messages. Data displayed are averages of three independent experiments using four different HFKs donor strains (n = 4 replicates). All mRNA levels were normalized to TBP. <b>B.</b> Southern blot analysis of whole cell DNA extracted from the same coelectroporated cells graphed in A. DNA was separated by agarose gel electrophoresis and probed with ³²P-labeled HPV18 genome 72 hours after DNA transfection. Prior to electrophoresis, DNA was digested with the restriction endonucleases DpnI to remove input, un-replicated DNA and EcoRI to linearize the viral genome. The arrow indicates the linear form of the viral DNA. Blot was performed using DNA from one of the two replicates shown in panel A. <b>C</b>. Phosphorimage quantitation of Southern blot assays from two independent experiments, except Sp100B-Q sample was tested only once. Error bars represent SEM. The paired student t-test was used to determine statistical significance between the EYFP-transfected control and each Sp100 isoform for panel A and C. n.s., not statistically significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p