Detection of endogenous KSHV present in oropharyngeal cells of WT KSHV donors and early-passage primary epithelial cell cultures. (A) Immunofluorescent confocal microscopic detection of KSHV antigens. Monolayers were fixed and incubated with isotype antibody or an antibody against KSHV LANA or glycoprotein K8.1. Secondary antibodies conjugated to AlexaFluor 488 were used to detect the primary antibody bound to the KSHV protein, and ToPro-3 dye was used to detect nuclear DNA. Panel 1, antigen-positive controls (TPA-induced BCBL1 cells). Upper photos show the Alexa 488 fluorescence, and lower photos show the nuclei stained with ToPro-3. Panel 2, donor oropharyngeal cells. Upper photos show the Alexa 488 fluorescence, and lower photos show the nuclei stained with ToPro-3. Panel 3, P-EPI-2 passage 4. Upper photos show the Alexa 488 fluorescence, and lower photos show the nuclei stained with ToPro-3 merged with the Alexa 488 fluorescence and the bright-field images. Panel 4, T-EPI 1 passage 4. Upper photos show the Alexa 488 fluorescence, and lower photos show the nuclei stained with ToPro-3 merged with the Alexa 488 fluorescence and the bright-field images. (B) Detection of KSHV ORF 26 by conventional PCR. DNAs from uninfected BJAB and latently infected BCBL1 cells were used as negative and positive controls, respectively. Lanes 1 to 7, oropharyngeal DNA isolated from donor TW samples; lane 8, P-EPI-2 passage 7 cell DNA; lane 9, BCBL1 cell DNA; lane 10, BJAB cell DNA; lane 11, no template DNA; lane 12, T-EPI 1 passage 6 DNA (this sample was run on another gel, but the PCRs were performed in the same experiment as the rest of the lanes). (C) Real-time PCR amplification plot of KSHV ORF 73 in donor oropharyngeal cell DNA. DNAs isolated from TW donor oropharyngeal cells from one KSHV-seropositive (sero⁺) donor and six other donors (TW donors) were used as templates for real-time PCR detection of KSHV ORF 73 DNA. DNA isolated from uninfected BJAB and latently infected BCBL1 cells served as negative and positive controls, respectively. KSHV DNA copy numbers relative to a standard curve of 10-fold serially diluted BCBL1 DNA are shown in parentheses. n. det., not detectable.

Detection of KSHV in epithelial cell lines infected with WT KSHV. (A) Detection of WT KSHV antigens in MeWo cells. Monolayers were fixed and incubated with antibodies against KSHV LANA or K8.1 glycoproteins at 24 h postinfection. Secondary antibodies conjugated to Alexa 488 were used to detect the primary antibody bound to the KSHV protein, and ToPro-3 dye was used to detect the cell nuclei. Upper photos show the Alexa 488 channel, and lower photos show the Cy5 channel and the ToPro-3-stained nuclei. (B) PCR detection of KSHV ORF 26 DNA in MeWo cells. Three hundred nanograms of cellular DNA was used as template for the first round PCR, and 5 μl of the first round reaction was use as template DNA for the second round. Lane 1, mock-infected cells; lane 2, 48 hpi BCBL1 virus-infected cells; lane 3, 48 hpi WT virus-infected cells; lanes 4 and 5, DNA isolated from BCBL1 or BJAB cells that was used as positive and negative controls, respectively; lane 6, no DNA. (C) Real-time PCR detection of WT KSHV ORF 73 DNA in MeWo cells. Three hundred nanograms of DNA isolated from infected with either BCBL1 virus or WT TW virus (WT virus) or mock-infected cells and harvested at 16 and 96 h postinfection was used as template for real-time PCR detection of KSHV ORF 73 DNA. DNA from TPA-activated BCBL1 cells served as a positive control. KSHV DNA copy numbers relative to a standard curve of 10-fold serially diluted BCBL1 DNA are in parentheses. n. det., not detectable. (D) Detection of WT KSHV antigens in A293T cells at 24 to 96 hpi. Infected monolayers were fixed and incubated with antibody against KSHV K8.1 glycoprotein. Secondary antibodies conjugated to AlexaFluor 488 were used to detect the primary antibody bound to the KSHV protein, and ToPro-3 dye was used to detect the cell nuclei.

Detection of WT KSHV infection of primary oral epithelial cells. P-EPI-2 passage 5 (A) and T-EPI 1 passage 10 (B) monolayers were fixed and incubated with isotype antibody, or antibodies against KSHV LANA or K8.1 glycoprotein at 24, 48, 72, and 98 hpi with virus isolated from throat wash samples from 4 different donors; one donor used for infections shown in Fig. 3A, and virus isolated from three other donors was used for panels B to E. Virus isolated from TPA-induced-BCBL1 and uninfected BJAB supernatant (Endog.) were used as positive and negative controls, respectively. (A) Expression kinetics of latent (LANA) and lytic (K8.1) KSHV antigens in P-EPI cells. Secondary antibodies conjugated to AlexaFluor 488 were used to detect the primary antibody bound to KSHV antigens, and ToPro-3 dye was used to detect the cell nuclei. Upper photos in each row show the AlexaFluor 488 channel, and lower photos show the AlexaFluor 488 and ToPro-3 and bright field. Row 1, 48 hpi K8.1 expression (left panels, isotype control); row 2, 48 hpi LANA expression; row 3, 72 hpi K8.1 expression; row 4, 72 hpi LANA expression. (B) Expression kinetics of latent (LANA) and lytic (K8.1) KSHV antigens in T-EPI cells. Secondary antibodies conjugated to AlexaFluor 488 were used to detect the primary antibody bound to KSHV antigens, and ToPro-3 dye was used to detect the cell nuclei. Upper photos in each row show the AlexaFluor 488 channel, and lower photos show the AlexaFluor 488, ToPro-3 and bright field, or AlexaFluor 547 overlay. Row 1, 24 hpi K8.1 expression; row 2, 24 hpi LANA expression; row 3, 96 hpi K8.1 expression; row 4, 96 hpi LANA expression. Secondary antibodies conjugated to AlexaFluor 488 were used to detect the primary antibody bound to KSHV antigens, and ToPro-3 dye was used to detect the cell nuclei. Upper photos in each row show the AlexaFluor 488 channel, and lower photos show the AlexaFluor 488, ToPro-3 and bright field, or AlexaFluor 547 overlay. (C) More lytic than latent antigen expression in T-EPI cells. Secondary antibodies conjugated to goat anti-rabbit AlexaFluor 547 (red) or goat anti-rat or mouse AlexaFluor 488 (green) were used to detect the primary antibody bound to KSHV antigens, and ToPro-3 dye (blue) was used to detect the cell nuclei. Upper panels show the AlexaFluor 488 and AlexaFluor 547 channels; and lower panels show the AlexaFluor 488, AlexaFluor 547, and ToPro-3 overlay. Left photos, negative and positive controls in BCBL1 cells. Center photos, faint LANA expression (red),cytoplasmic K8.1 (red) expression, and DNA (blue) in T-EPI cells 38 hpi with WT KSHV. Right photos, cytoplasmic gB (red) and perinuclear LANA expression (green). (D) Detection of K8.1 cDNA in P-EPI cells. cDNA generated from RNA isolated from 10⁵ PEPI cells at 96 hpi was used as template for real-time PCR amplification of a 161-bp region of the K8.1 gene containing a 94-bp splice (19). The PCR was electrophoresed on a 2% agarose gel. A ³²P-labeled oligonucleotide probe internal to the primer pair was hybridized to the amplification products after Southern blot transfer. Induced BCBL1 cells served as positive controls. Duplicate cDNA samples generated with (RT⁺ lanes; 161-bp product from spliced template) and without added reverse transcriptase (RT⁻ lanes; 255-bp product from unspliced template) served as controls to detect genomic DNA. (E) Real-time PCR detection of KSHV DNA in passage 7 T-EPI 1-infected cells. DNA was isolated from cells infected with BCBL virus or WT TW virus (WT) or mock infected. Cells harvested at 2 days postinfection (top panel) or after 1 passage at 8 days postinfection (bottom panel) were used as templates for real-time PCR detection of KSHV ORF 73 DNA. DNA from TPA-activated BCBL1 cells served as a positive control (upper panel). Endog, DNA from an untreated low passage T-EPI cell monolayer containing endogenous virus. KSHV DNA copy numbers relative to a standard curve of 10-fold serially diluted BCBL1 DNA are in parentheses. n. det., not detectable.

Representative scanning electron micrograph of KSHV virions (blue or white) at ×40,000 magnification on infected P-EPI cells (epithelial cell plasma membrane extensions shown in green). P-EPI cells infected with either WT or BCBL1 KSHV were cultured for 48 h prior to processing.

SEM detection of KSHV virions on P-EPI cell membranes. Endogenously infected P-EPI-3 passage 3 cells (E and F) were infected with WT KSHV (C and D) or control BCBL1 (A and B) virus and cultured for 48 h prior to processing. Magnification: ×15,000 for panels A, C, and E; ×5,000 for panels B, D, and F.

Inhibition of WT KSHV replication or infection in epithelial cells. (A) Inhibition of lytic replication in primary epithelial cells by ganciclovir. P-EPI-2 passage 5 cells were infected with WT KSHV or control BCBL1 virus and cultured with or without 2.5 mg of ganciclovir/ml for 24 h prior to IFA detection of glycoprotein K8.1. Left panel, no ganciclovir; right panel, ganciclovir added. Upper photos show the Alexa 488 filter, and lower photos show the Alexa 488 and ToPro-3 and bright-field overlay. (B) Inhibition of WT KSHV lytic replication in A293T cells by acyclovir. A293T cells were infected in the presence of 10 mg of acyclovir/ml, and examined at 48 hpi. Upper photos show the Alexa 488 channel, and lower photos show the Alexa 488, ToPro-3 and bright-field overlay. (C) Inhibition of WT KSHV infectivity by neutralizing antibody against gB. Virus suspension was incubated with 60μg of anti-gB or negative control UK218 antibody/ml for 1 h prior to infection of T-EPI or A293T cells. At 96 hpi, cells were processed for IFA. Photos show the Alexa 488 and ToPro-3 overlay. (D) Ganciclovir-mediated inhibition of lytic gene expression in A293T cells. A293T cells were infected in the presence or absence of 2.5-mg/ml ganciclovir (± GAC) for 24 h (upper panel) or 72 h (lower panel). cDNA with or without reverse transcriptase (RT±) was generated from isolated RNA and used as template for real-time PCR amplification of a region of the K8.1 gene containing a 94-bp splice (19). The PCR was electrophoresed on a 2% agarose gel. A ³²P-labeled oligonucleotide probe internal to the primer pair was hybridized to the amplification products after Southern blot transfer. Induced BCBL1 cells served as positive controls. Duplicate cDNA samples generated with (RT⁺ lanes; 161-bp product from spliced template) and without added reverse transcriptase (RT⁻ lanes; 255-bp product from unspliced template) served as controls to detect genomic DNA.

Model of KSHV infection and reactivation in the oral cavity of immunocompetent individuals. Lytic replication in the oral epithelium may serve as a reservoir of infectious virus. (A) K8.1-positive oral epithelial cell. Virus from this reservoir may then infect trafficking lymphocytes and lymphoid tissue in the oropharynx, replicate and then establish latency. (B) K8.1-positive tonsil cell 24 hpi with TW-derived virus. This may in turn provide a latent virus reservoir. Purple circles in nuclei represent latently infected cells.