Jennifer Berline

Epstein-Barr virus infection of polarized tongue and nasopharyngeal epithelial cells

Epstein-Barr virus (EBV) initially enters the body through the oropharyngeal mucosa and subsequently infects B lymphocytes through their CD21 (CR2) complement receptor. Mechanisms of EBV entry into and release from epithelial cells are poorly understood. To study EBV infection in mucosal oropharyngeal epithelial cells, we established human polarized tongue and pharyngeal epithelial cells in culture. We show that EBV enters these cells through three CD21-independent pathways: (i) by direct cell-to-cell contact of apical cell membranes with EBV-infected lymphocytes; (ii) by entry of cell-free virions through basolateral membranes, mediated in part through an interaction between b1 or α5β1 integrins and the EBV BMRF-2 protein; and (iii) after initial infection, by virus spread directly across lateral membranes to adjacent epithelial cells. Release of progeny virions from polarized cells occurs from both their apical and basolateral membranes. These data indicate that multiple approaches to prevention of epithelial infection with EBV will be necessary.

Oxidation of methionine 63 and 83 regulates the effect of S100A9 on the migration of neutrophils in vitro

The calcium‐binding proteins S100A8 and S100A9 and their heterocomplex calprotectin are abundant cytosolic constituents in human neutrophils, constitutively expressed by mucosal epithelium and in association with inflammation by epidermal keratinocytes. S100A8 and S100A9 are pleiotropic proteins, which partake in the regulation of leukocyte migration. This study was designed to investigate the effect of S100A9 on neutrophil migration and to explore the mechanisms that regulate this effect. Based on previous results with S100A8, we hypothesized that S100A9 repels neutrophils and that oxidation of S100A9 regulates this function. Using standard Transwell chemotaxis assays and site‐directed mutagenesis, we show that S100A9 exerts a chemo‐repulsive (fugetactic) effect on peripheral neutrophils, an effect abolished by oxidation of S100A9. After substitution of methionine 63 and 83 for alanine, S100A9 maintained its fugetaxis activity, even in inhibitory, oxidative conditions. Together, the data suggest that S100A9 serves as a molecular switch for oxidative control of inflammation regulated by the oxidation of species‐conserved methionine residues. In healthy mucosal tissue, expression of S100A9 by the epithelium may serve to inhibit leukocyte recruitment. However, conditions of oxidative stress, including infection and overgrowth of opportunistic pathogens, may abrogate this activity by neutralizing S100A9 as a result of its oxidative alteration.

Inhibition of Human Papillomavirus Type 16 E7 Phosphorylation by the S100 MRP-8/14 Protein Complex

ABSTRACT The human papillomavirus type 16 (HPV16) E7 is a major viral oncoprotein that is phosphorylated by casein kinase II (CKII). Two S100 family calcium-binding proteins, macrophage inhibitory-related factor protein 8 (MRP-8) and MRP-14, form a protein complex, MRP-8/14, that inactivates CKII. The MRP-8/14 protein complex may inhibit CKII-mediated E7 phosphorylation and therefore may alter its interaction with cellular ligands and reduce E7 oncogenic activity. We examined the inhibitory effect of the MRP-8/14 complex on CKII activity and HPV16 E7 phosphorylation. We have shown that CKII activity and HPV16 E7 phosphorylation were inhibited by uptake of exogenous MRP-8/14 and activation of endogenous MRP-8/14. MRP-8/14-mediated inhibition of E7 phosphorylation occurred at the G1 phase of the cell cycle. Analysis of MRP expression in primary keratinocytes and in HPV16- and 18-transformed cervical and foreskin epithelial cell lines showed that expression of MRP-8, MRP-14, and the MRP-8/14 complex was detected only in primary untransformed keratinocytes and not in the HPV-infected immortalized epithelial cells. CKII activity in HPV-immortalized keratinocytes was approximately fourfold higher than in HPV-negative primary keratinocytes. Treatment of HPV-positive immortalized epithelial cells with exogenous MRP-8/14 resulted in E7 hypophosphorylation and complete inhibition of cell growth within 2 weeks, compared with HPV-negative primary and immortalized HPV-negative cervical epithelial cells, which showed 25 and 40% growth inhibition, respectively. Together these results suggests that the MRP-8/14 protein complex in HPV-infected epithelial cells may play an important role in regulation of CKII-mediated E7 phosphorylation and inhibition of its oncogenic activity.

S100A8 Triggers Oxidation-sensitive Repulsion of Neutrophils

The inflammatory response to tissue injury is a multi-faceted process. During this process, neutrophils migrate in the extravascular spaces, directed to the site of injury by chemical gradients generated by chemotactic molecules. S100A8, a protein associated with a wide variety of inflammatory conditions, is heavily over-expressed in association with inflammation. We hypothesized that human S100A8 possesses neutrophil-repelling properties that result in an anti-inflammatory effect in vivo. The chemotactic activity of S100A8 on neutrophils was tested in Transwell chemotaxis assays. Analysis of the data indicates that S100A8 causes a repulsion of peripheral neutrophils, an activity that S100A8 loses upon its oxidation. Using a mutant of S100A8 resistant to oxidation and consistent with the in vitro findings, we demonstrated that S100A8 causes a strong anti-inflammatory effect in the rat air-pouch model of inflammation in vivo. These data highlight a naturally occurring novel anti-inflammatory pathway and provide potential molecular targets for the development of novel anti-inflammatory therapeutics. Abbrevations: ethylene diamine tetraacetic acid (EDTA); limulus amoebocyte lysate assay (LAL); pertussis toxin (PTX); forward scatter (FSC); Interleukin-8 (IL-8); formyl-Met-Leu-Phe (fMLP); monocyte chemotactic protein 1 (MCP1).

Evidence for trafficking of Epstein-Barr virus strains between hairy leukoplakia and peripheral blood lymphocytes.

Hairy leukoplakia (HL), an epithelial lesion found on the side of the tongue in immunocompromised individuals, is characterized by high-level replication of Epstein-Barr virus (EBV) and multiple EBV strains. The source of these strains and their relationship to peripheral blood lymphocyte (PBL) strains has not previously been characterized. Using matched pairs of HL scrapings and PBL from 16 HIV-positive men, variation in EBV strain identity was characterized by detection of a 30 nucleotide deletion of the EBV latent membrane protein (LMP)-1 gene, variation in the LMP-1 repeat region and typing for Epstein-Barr nuclear antigen (EBNA)-2. Multiple EBV strains were found in both the HL and PBL specimens, but 13 of 16 (81%) patients showed evidence of strain identity for at least one strain and analysis of two patients suggested that EBV strains from HL could infect the PBL. Our data are consistent with active trafficking of EBV between these two compartments.

EBV BMRF-2 facilitates cell-to-cell spread of virus within polarized oral epithelial cells.

We previously reported that the Epstein-Barr virus (EBV) BMRF-2 protein plays an important role in EBV infection of polarized oral epithelial cells by interacting with beta1 and alphav family integrins. Here we show that infection of polarized oral epithelial cells with B27-BMRF-2(low) recombinant virus, expressing a low level of BMRF-2, resulted in significantly smaller plaques compared with infection by parental B95-8 virus. BMRF-2 localized in the trans-Golgi network (TGN) and basolateral sorting vesicles and was transported to the basolateral membranes of polarized epithelial cells. Mutation of the tyrosine- and dileucine-containing basolateral sorting signal, YLLV, in the cytoplasmic domain of BMRF-2 led to the failure of its accumulation in the TGN and its basolateral transport. These data show that BMRF-2 may play an important role in promoting the spread of EBV progeny virions through lateral membranes of oral epithelial cells.

EXPRESSION OF EPSTEIN-BARR VIRUS BMRF-2 AND BDLF-3 GENES IN HAIRY LEUKOPLAKIA

The high level of Epstein-Barr virus (EBV) replication found in hairy leukoplakia (HL) provides a unique opportunity to study EBV expression in the oral epithelium. Screening of a cDNA library from an HL biopsy revealed expression of two genes not previously described in vivo: BMRF-2 and BDLF-3. Sequence analysis of the cDNAs demonstrated several nucleotide changes from the B95-8 sequence. In all six different HL strains studied, only one amino acid change was found in BMRF-2 relative to B95-8 and two amino acid changes were found in the BDLF-3 ORF. mRNA expression of both genes was localized to the lower prickle cell layer of the tongue epithelium. BMRF-2 protein expression was primarily detected in the cell nuclei of the upper prickle cell layer; immunoelectron microscopy revealed that BMRF-2 was associated with the nuclear chromatin. BDLF-3 protein expression was observed in the perinuclear space and cytoplasm of the prickle cells. BDLF-3 has recently been identified as a virion-associated protein, but the functions of BMRF-2 and BDLF-3 have not been elucidated.