Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures - PubMed (original) (raw)

Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures

D M Ciufo et al. J Virol. 2001 Jun.

Abstract

Angiogenic Kaposi's sarcoma (KS) skin lesions found in both AIDS and non-AIDS patients are universally associated with infection by the presumed causative agent, known as KS-associated herpesvirus (KSHV) or human herpesvirus 8. KSHV genomes expressing latent state virus-encoded mRNAs and the LANA1 (latent nuclear antigen 1) protein are consistently present in spindle-like tumor cells that are thought to be of endothelial cell origin. Although the KSHV lytic cycle can be induced in rare latently infected primary effusion lymphoma (PEL) cell lines, the ability to transmit or assay infectious KSHV has so far eluded investigators. Here, we demonstrate that infection with supernatant virions derived from three different tetradecanoyl phorbol acetate-induced PEL cell lines can induce cultured primary human dermal microvascular endothelial cells (DMVEC) to form colonies of proliferating latently infected spindle-shaped cells, all of which express the KSHV-encoded LANA1 protein. Although their initial infectivity varied widely (JSC1 > > BC3 > BCP1), virions from all three cell lines produced distinctive spindle cell colonies and plaques without affecting the contact-inhibited cobblestone-like phenotype of adjacent uninfected DMVEC. Each infected culture could also be expanded into a completely spindloid persistently infected culture displaying aggregated swirls of spindle cells resembling those in KS lesions. Formation of new colonies and plaques was inhibited in the presence of phosphonoacetic acid or gangciclovir, but these antiherpesvirus agents had little effect on the propagation of already latently infected spindloid cultures. In persistently infected secondary cultures, patches of up to 10% of the spindloid cells constitutively expressed several early viral lytic cycle proteins, and 1 to 2% of the cells also formed typical herpesvirus DNA replication compartments, displayed cytopathic rounding effects, and expressed late viral antigens. We conclude that de novo KSHV infection induces a spindle cell conversion phenotype in primary DMVEC cultures that is directly associated with latent state expression of the LANA1 protein. However, these cultures also spontaneously reactivate to produce an unusual combination of both latent and productive but slow lytic cycle infection. The formation of spindle cell colonies and plaques in DMVEC cultures provides for the first time a quantitative assay for directly measuring the infectivity of KSHV virion preparations.

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Figures

FIG. 1

KSHV infection of contact-inhibited DMVEC cultures produces either colonies and plaques or total conversion to an elongated spindle cell phenotype. (A) Plaque dish assay for infectious KSHV in primary human DMVEC monolayer cultures showing four individual spindle cell colonies (circled) that developed 25 days after infection with a 100-μl sample of pelleted filtered supernatant virions from TPA-treated BC3 PEL cells. The two largest colonies contain large central patches showing CPE (= plaques). (Crystal violet stain.) (B) Medium-power photomicrograph of the top edge of one of the two smaller BC3 colonies from panel A, containing several hundred spindle cells and showing the boundaries of the colony with adjacent cobblestone-like areas of uninfected DMVEC. (Crystal violet stain; 16× objective.) (C) Control cobblestone-like monolayer of cuboidal contact-inhibited DMVEC 20 days after addition of 100 μl of resuspended pelleted material from the filtered supernatant of untreated JSC1 cells. (Crystal violet stain; 16× objective.) (D) Parallel culture of completely, spindle cell-converted DMVEC photographed at 20 days after receiving 100 μl of resuspended pelleted KSHV virions from the filtered supernatant of TPA-treated JSCI cells. (Crystal violet stain; 16× objective.)

FIG. 2

The LANA1 protein is expressed in all spindle cell-Converted KSHV-infected DMVEC. (A) High-power phase-contrast photomicrographs of converted spindle cells in a secondary JSC1-infected DMVEC culture at 7 days after 1:10 dilution with fresh uninfected DMVEC. (Crystal violet stain; 40× objective.) (B) Expression of the LANA1 latency protein (brown nuclei) in all spindle cells within two distinct 30- to 80-cell colonies that had formed in a primary infected DMVEC culture at 17 days after addition of JSC1 supernatant virus. LANA1 was detected by IHC with rat anti-LANA1 MAb and peroxidase-DAB chromogen (40× objective.) (C) High-power phase-contrast photomicrograph of a partially spindle cell-converted second-passage DMVEC culture at 24 days after addition of filtered supernatant from a fresh JSC1-infected DMVEC spindle cell culture showing a latently infected spindle cell colony containing a small patch or plaque of rounded cells with typical herpesvirus CPE and some remaining uninfected cuboidal cells. (Crystal violet stain; 40× objective.) (D) Double-label IHC detection of both latent and late lytic cycle KSHV-encoded proteins in a completely spindle cell-converted JSC1-infected DMVEC culture at 3 days after reseeding in a slide chamber dish. Brown chromogen, LANA1 nuclear staining in all latently infected spindle cells (rat anti-LANA1 MAb; peroxidase and DAB). Red/pink chromogen, late lytic cytoplasmic gpK8.1 membrane protein in the subfraction of cells showing rounding and CPE (mouse MAb; phosphatase and Vector red; 40× objective.)

FIG. 3

Presence of punctate LANA1 patterns, loss of von Willebrand factor positivity, and spontaneous reactivation of early lytic viral protein expression in persistently infected DMVEC spindle cell cultures. (A and B) High-power photomicrographs of punctate nuclear LANA1 staining patterns in JSC1-infected DMVEC spindle cells. (A) IFA with rabbit anti-LANA1 PAb green (fluorescein isothiocyanate [FITC] label; 63× objective) in a persistently infected secondary spindle cell culture; (B) IHC with rat anti-LANA1 MAb and peroxidase-DAB (brown; 100× objective) in a 16-day primary spindle cell colony. (C and D) Expression of von Willebrand factor and LANA1 detected by double-label IFA in partially JSC1-infected DMVEC cell culture. (C) Mouse anti-VWF MAb (FITC, green cytoplasm bodies); (D) rabbit anti-LANA1 PAb (rhodamine label, red nuclei) in the same field (63× objective). (E) Expression of KSHV-encoded early lytic cycle ORF-K8 nuclear protein in sporadic spindle cells in a JSCS-infected DMVEC spindle cell culture detected by IFA with rabbit anti-K8 PAb (green FITC label; 40× objective). (F) Expression of the KSHV-encoded early lytic cycle cytoplasmic membrane protein ZMP-A(K5) in sporadic spindle cells in a JSC1-infected DMVEC spindle cell culture as detected by IFA with rabbit anti-ZMP-A PAb (green FITC label; 40× objective).

FIG. 4

Spontaneous lytic cycle reactivation demonstrated by IHC in persistently KSHV-infected DMVEC spindle cell cultures. (A and B) Low-power (16× objective) and high-power (40× objective) views of KSHV-encoded early cytoplasmic membrane protein ZMP-A expressed in a subset of the spindle cells in a completely converted JSC1-infected DMVEC spindle culture at 3 days after reseeding in slide chambers, as detected by IHC with rabbit anti-ZMPA PAb (phosphatase and Vector red chromogen). (C and D) Low-power (16× objective) and high-power (40× objective) views of KSHV-encoded ORF-K8 early nuclear protein expressed in a subset of the spindle cells in a completely converted JSC1-infected DMVEC spindle cell culture at 3 days after reseeding in slide chambers, as detected by IHC with rabbit anti-ORFK8 PAb (phosphatase and Vector red chromogen).

FIG. 5

Development of clusters of infected DMVEC displaying late lytic CPE and containing active viral DNA replication compartments. (A) Expression of the KSHV-encoded ORF59 protein including typical late stage herpesvirus nuclear DNA replication compartments within a clustered miniplaque in a 10-day confluent secondary JSC1 spindle cell culture, as detected by IFA with mouse anti-ORF59 MAb (red rhodamine label, 63× objective). (B) Expression of ORF-K8 including nuclear DNA replication compartment-associated forms within a clustered miniplaque in a 10-day confluent secondary JSC1 spindle cell culture, as detected by IFA with rabbit anti-K8 PAb (green fluorescein isothiocyanate [FITC] label; 63× objective). (C and D) Expression of the KSHV-encoded lytic membrane protein ZMP-A in small plaque-like patches of rounded late stage lytic cycle-infected DMVEC cells displaying CPE. Two different fields within a 10-day secondary JSC1 spindle cell culture detected by IFA with rabbit anti-ZMP-A PAb (green FITC label; 63× objective) are shown. (E and F) Colocalization of ORF59 and ORF-K8 by double-label IFA in viral DNA replication compartments in a late lytic stage JSCI-infected DMVEC cell, showing rounding and CPE, as detected with mouse anti-ORF59 MAb (E; red rhodamine label) and rabbit anti-ORF8 PAb (F; green FITC label) in the same field (100× objective). (G and H) Colocalization of newly synthesized DNA (BUdR pulse-label) and the ORF-K8 nuclear protein in a mature viral DNA replication compartment in a late lytic stage rounded JSC1-infected DMVEC cell as detected by double-label IFA using mouse anti-BUdR MAb (G; red rhodamine label) and rabbit anti-K8 PAb (H; green FITC label) in the same field (100× objective).

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Spindle cell conversion by Kaposi's sarcoma-associated herpesvirus: formation of colonies and plaques with mixed lytic and latent gene expression in infected primary dermal microvascular endothelial cell cultures - PubMed (original) (raw)