Role of adherens junction proteins in differential herpes simplex virus type 2 infectivity in communication-competent and -deficient cell lines.

BACKGROUND: Gap junctional intercellular communication decreases with HSV-2 infection. To determine the importance of functional gap junctions for infectivity, we compared HSV-2 growth in communication-competent and -deficient cell lines. METHODS: HSV-2 infectivity was tested in five cell lines: WB rat liver epithelial cells (communication-competent), WB-aB1 (communication-deficient), WB-a/32-10 (communication-rescued), HeLa (communication-deficient), and Cx43-transfected HeLa (communication-rescued) cells. HSV-2 growth curves and indirect immunofluorescence labeling of viral and cell proteins were performed in wild-type and mutant WB cells. RESULTS: Although wild-type WB cells were highly permissive for HSV-2 infection, virus production was significantly attenuated in communication-deficient and -rescued mutant WB cells. HeLa exhibited no difference in infectivity between communication-competent and -deficient cell lines. Tight and adherens junction proteins, including zonula occludens-1 and nectin-1, were not different in the WB cell lines. However, E-cadherin levels were elevated and ß-catenin was found to co-localize with glycoprotein E, a viral glycoprotein associated with cell-to-cell spread, in the mutant WB cells. CONCLUSIONS: These results suggest that attenuated viral production in mutant WB cells is due to viral protein co-localization with adherens junction proteins rather than the loss or restoration of functional gap junctions.

Herpes simplex virus-type 2 infectivity and agents that block gap junctional intercellular communication.

In rat liver epithelial (WB) cells, the protein kinase C inhibitor H7 blocked gap junctional intercellular communication (GJIC) and reduced virus infectivity. Octanol, 18-beta-glycyrrhetinic acid, and staurosporine, agents that reduce GJIC, had no effect upon virus infectivity. Previous studies demonstrated that herpes simplex virus-type 2 (HSV-2) infection was accompanied by attenuated GJIC. Of agents tested, only H7 reduced plaque forming unit (pfu) ability in a dose-dependent manner with 100% plaque reduction at 40 microM without evidence of cytotoxicity. Dye transfer indicated that H7 decreased GJIC, although Western blotting revealed that it did not alter phosphorylation of the gap junction protein, connexin 43 (Cx43). Using indirect immunofluorescence, Cx43 was found to localize in membrane plaques in uninfected cells and H7 did not alter this distribution. However, Cx43 was lost from the membrane at 24h in both H7-treated and untreated cells infected with HSV-2. Viral infection increased serine phosphorylation, particularly in the nuclear region, and this effect was reduced following H7 treatment. Thus, H7 attenuated both GJIC and infectivity of HSV-2 in WB cells but the anti-viral effects were due to reduced nuclear protein phosphorylation rather than alterations in phosphorylation or localization of Cx43.

Antiviral agents alter ability of HSV-2 to disrupt gap junctional intercellular communication between mammalian cells in vitro.

In cultured mammalian cells (Vero), different antiviral agents change to differing degrees the ability of HSV2 to down-regulate gap junctions, each agent having a specific effect. Measured by intracellular electrodes, control cell populations showed 49-51% coupling, uninfected populations treated with acyclovir or SDS averaged 43-51% coupling while populations infected with HSV2 had coupling reduced to 8%. The antiviral agent acyclovir (1 microg/ml), which suppresses viral replication, failed to prevent this down regulation (final coupling ratio of 11%). A plant extract (250 microg/ml) from Pilostigma thonningii offered slightly more protection (final coupling ratio of 22%), while sodium dodecyl sulfate (SDS) (50 microM) afforded nearly complete protection (final coupling ratio of 40%). With SDS there was an initial down regulation to only 16% coupling by 3 h post infection, followed by a recovery of intercellular communication to near control levels by 24 h. While SDS was originally believed to alter the viral coat and prevent entry into the cell, our data are in agreement with recent studies which indicate that SDS treated viruses can enter into host cells, but in a severely diminished condition. Our data also suggest that the gap junction antagonist is brought into the cells as part of the entering virus.

LC-MS/MS method for confirmation of recombinant human erythropoietin and darbepoetin alpha in equine plasma.

Recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production. Consequently, they are abused in human and equine sports. To deter their abuse in the horse racing industry, a sensitive and reliable method for confirmation of these agents in equine plasma has been in urgent need. Such a method by LC-MS/MS is described in this paper. The method involved analyte enrichment by immunoaffinity separation using anti-rhEPO antibody linked to magnetic beads, digestion by trypsin, and analysis by LC-MS/MS. Two specific proteotypic peptides, 46VNFYAWK52 and 144VYSNFLR150 from rhEPO and DPO were employed for confirmation of the analytes based on chromatographic retention times and major product ions. The limit of confirmation of this method was 0.2 ng/mL, and the limit of detection was 0.1 ng/mL for rhEPO and DPO in equine plasma. This method was successful in confirming the presence of rhEPO and DPO in plasma samples collected from research horses to which rhEPO or DPO was administered and from racehorses following competition and in noncompetition samples in North America. To our knowledge, this is the first LC-MS method with adequate sensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples. This method provides a powerful enforcement tool that was lacking in the fight against the abuse of rhEPO and DPO in the horse racing industry.