Figure 1.

Reduced CD31 expression on KSHV infected E-DMVECs correlates with spontaneous or PMA-induced expression of K5. (A) Flow cytometry of CD31 and K5 expression in uninfected (E-DMVEC) or infected cells (E-DMVEC KSHV) treated with PMA (dark line, no shading) or untreated (gray shading) for 48 hours. Cell-surface–expressed CD31 was detected on intact cells using monoclonal antibody JC/704; the dotted line indicates fluorescently labeled secondary antibody only. To reveal K5 expression, KSHV-infected cells or uninfected E-DMVECs (dotted line) were permeabilized and stained with K5-specific antibody 328C7 as well as fluorescently labeled secondary antibody. (B) Flow cytometry of CD31 or K5 expression of uninfected primary DMVECs () or primary DMVECs 48 hours after infection with KSHV (dark line, no shading). (C) Spontaneous K5 expression in latently infected E-DMVECs in the absence of PMA. Left column shows KSHV-infected E-DMVECs stained for CD31 (green), K5 (red), and LANA-1 (red). LANA-1 staining is evident by a typical punctate nuclear pattern. Right column shows staining as in the left column, but without antibodies to LANA-1. The arrow indicates a K5-expressing cell. Note the lack of CD31 expression on K5-expressing cells, whereas LANA-expressing, but K5-negative, cells still express CD31. (D) Simultaneous evaluation of K5 and ORF59 in KSHV-infected DMVECs after PMA induction (48 hours). Monolayers were stained for expression of K5 and ORF59 using specific antibodies and isotype-specific secondary antibodies (Alexa 488 IgG₁ for ORF59 and Alexa 594 IgG_2b for K5). All cells expressing the IE protein K5 expressed the E protein Orf59, but 4 to 5 times as many cells expressed K5 only. Similar ratios, but lower absolute numbers, were observed in the absence of PMA (not shown).