Activation of Kaposi's sarcoma-associated herpesvirus lytic gene expression during epithelial differentiation

Abstract

The oral cavity has been identified as the major site for the shedding of infectious Kaposi's sarcoma-associated herpesvirus (KSHV). While KSHV DNA is frequently detected in the saliva of KSHV seropositive persons, it does not appear to replicate in salivary glands. Some viruses employ the process of epithelial differentiation for productive viral replication. To test if KSHV utilizes the differentiation of oral epithelium as a mechanism for the activation of lytic replication and virus production, we developed an organotypic raft culture model of epithelium using keratinocytes from human tonsils. This system produced a nonkeratinized stratified squamous oral epithelium in vitro, as demonstrated by the presence of nucleated cells at the apical surface; the expression of involucrin and keratins 6, 13, 14, and 19; and the absence of keratin 1. The activation of KSHV lytic-gene expression was examined in this system using rKSHV.219, a recombinant virus that expresses the green fluorescent protein during latency from the cellular EF-1alpha promoter and the red fluorescent protein (RFP) during lytic replication from the viral early PAN promoter. Infection of keratinocytes with rKSHV.219 resulted in latent infection; however, when these keratinocytes differentiated into a multilayered epithelium, lytic cycle activation of rKSHV.219 occurred, as evidenced by RFP expression, the expression of the late virion protein open reading frame K8.1, and the production of infectious rKSHV.219 at the epithelial surface. These findings demonstrate that KSHV lytic activation occurs as keratinocytes differentiate into a mature epithelium, and it may be responsible for the presence of infectious KSHV in saliva.

FIG. 1.

Characterization of in vitro organotypic raft culture epithelium by immunofluorescence histochemistry in cross section. Monoclonal antibody detection of selected keratins (13, 1, 14, 6, and 19) and involucrin (INV) is indicated for each row on the left-hand side. For each cross section, the following are shown: left column, phase; middle column, fluorescence for DAPI nuclear staining (blue); right column, computer-combined image of fluorescence for DAPI nuclear staining (blue, from the middle column) and Qdot 655-labeled antibody detection of the antibody reactive to the respective cellular protein in each row (red).

FIG. 2.

(A) Photomicrograph of undifferentiated tonsil keratinocytes 10 days postinfection with rKSHV.219 imaged for (1) phase, (2) GFP (green), and (3) RFP (red). (B) Detection of LANA in rKSHV.219-infected keratinocytes. (1) Phase image; (2) computer-combined image of fluorescence for GFP (green), DAPI nuclear staining (blue), and Alexa 647-labeled antibody detection of LANA.

FIG. 3.

Time course photomicrographs of intact organotypic raft cultures initiated with keratinocytes infected with rKSHV.219. (A) One day prior to air exposure. (B) Three days post-air exposure. (C) Six days post-air exposure. (D) Uninfected organotypic raft culture control 6 days post-air exposure. Left column, phase; middle column, fluorescence for GFP (green); right column, fluorescence for RFP.

FIG. 4.

Detection of ORF K8.1 protein in intact epithelial raft cultures (overhead view) 6 days post-air exposure. (A) Fluorescence for GFP (green). (B) Fluorescence for RFP (red). (C) Fluorescence for Qdot 655-labeled antibody detection of the anti-K8.1 monoclonal antibody (blue). (D) Computer-combined images of fluorescence for GFP (from pane A), RFP (from panel B), and K8.1 (from panel C).

FIG. 5.

Photomicrograph of an rKSHV.219-infected organotypic raft culture in cross section 6 days postairlifting. (A) Phase; (B) GFP (green); (C) RFP (red); (D) Computer-combined images of fluorescence for DAPI nuclear staining (blue), GFP (green), and RFP (red).

FIG. 6.

Detection of the late viral envelope glycoprotein K8.1 in cross sections of mature rKSHV.219-infected organotypic raft cultures. Shown are three representative computer-combined photomicrographs of fluorescence for DAPI nuclear staining (blue) and Qdot 655-labeled antibody detection of the anti-K8.1 monoclonal antibody (red).

FIG. 7.

Phase and fluorescence (GFP) photomicrographs of 293 cells 2 days postinfection with cell supernatant collected from the apical surfaces of mature organotypic raft cultures infected with rKSHV.219.