FIG 10.

Alteration of Cx43 in mouse brain due to MHV-A59 infection. Mice were infected intracranially with MHV-A59 at 50% of the LD₅₀ or with PBS-BSA for mock-infected mice. Mice were sacrificed at day 5 p.i., after which their liver and brain tissues were processed for RNA extraction. cDNA was synthesized from RNA of the brains and livers. To confirm infection, cDNAs from liver were amplified for virus-specific antinucleocapsid primers (IZJ5 and IZJ6). (A) Intracranial injection of mice with the virus showed the presence of nucleocapsid-specific amplicons (Infected lanes 1, 2, and 3) in liver. As expected, no such amplification was noted from mock-inoculated mice (Mock lanes 1 and 2). (B) Real-time qPCR analysis of the RNA samples from brain showed a significant 3.13- ± 0.06-fold reduction in relative abundance of Cx43 mRNA after MHV-A59 infection. The mean ± SEM incidences from three different mice are shown. (****, P < 0.0001; n = 3). Total protein was extracted from brain, and 20 μg of protein was resolved in SDS-PAGE, transferred to a PVDF membrane, and probed for Cx43 or the internal control, γ-actin. (C) Following infection with MHV-A59, the total Cx43 expression level was reduced, with a significant reduction observed for both 44- and 46-kDa isoforms of Cx43. This alteration was specific for Cx43, as GFAP expression was not changed significantly upon virus infection. Infection in mouse brains was confirmed by the presence of viral nucleocapsid protein (data not shown). (D) The nonphosphorylated form of Cx43 (42 kDa) expression was reduced 20.1% after MHV-A59 infection, whereas a 43.2% reduction was observed in phosphorylated forms of Cx43 (44 kDa and 46 kDa) after viral infection. The mean ± SEM incidences from three different animals is shown (*, P < 0.05).