FIG 4.

Intracellular localization of Cx43 in MHV-A59-infected primary astrocytes at an MOI of 2. Primary astrocytes were mock infected (A, C, and E) or infected with MHV-A59 at an MOI of 2 (B, D, and F). Cells were subjected to double-label immunofluorescence at 12 h (A and B), 24 h (C and D), and 36 h (E and F) p.i. with anti-Cx43 antisera (red) and anti-N antisera (green). Cells were visualized at 40× on a laser-scanning microscope. (A, C, and E) For the mock-infected cells, Cx43 was localized at the cell surface (thin arrow) with very minimal distribution in the intracellular compartment. In contrast, in MHV-A59-infected cells Cx43 was localized primarily in the intracellular compartment with very minimal distribution at the cell surface and was mostly colocalized with anti-N antisera (B, D, and E, thick arrow). Interestingly, intracellular retention of Cx43 was restricted to infected cells only. Cells negative for viral-N (B and D, thin arrow) did not show retention of Cx43 in an intracellular compartment. At each time point p.i., mock-infected cells (A, C, and E) expressed Cx43 at the cell surface (thin arrow), whereas Cx43 was localized mainly in the perinuclear compartment (B, D, and F, thick arrow) and partially colocalized with viral N protein in infected cells. To better illustrate these observed localization patterns, a single cell from the confluent astrocyte monolayer is shown at higher magnification in an inset.