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. 2001 Mar;75(6):2938-45.
doi: 10.1128/JVI.75.6.2938-2945.2001.

Internal ribosome entry site regulates translation of Kaposi's sarcoma-associated herpesvirus FLICE inhibitory protein

W Low  1 , M Harries, H Ye, M Q Du, C Boshoff, M Collins
Affiliations

Internal ribosome entry site regulates translation of Kaposi's sarcoma-associated herpesvirus FLICE inhibitory protein

W Low et al. J Virol. 2001 Mar.

Abstract

The gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) (or human herpesvirus 8) is associated with the endothelial tumor Kaposi's sarcoma (KS) and lymphoproliferative disorders in immunocompromised individuals. Only a small number of viral proteins are expressed in B cells latently infected with KSHV; here we characterize the mechanism of expression of one of these, the viral FLICE inhibitory protein v-FLIP (K13, ORF71). The v-FLIP coding region is present in a bicistronic message, following the v-cyclin coding region. Using both in vitro translation and cell transfection assays, we have identified an internal ribosome entry site (IRES) preceding the v-FLIP start codon and overlapping the v-cyclin (ORF 72) coding region, which allows v-FLIP translation. Using an antibody against v-FLIP we have detected expression of the endogenous protein in latently infected KSHV-positive primary effusion lymphoma (PEL) cell lines. Induction of apoptosis by serum withdrawal from PEL cells results in a relative increase in v-FLIP synthesis, as previously described for some cellular proteins translated from IRES.

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Figures

FIG. 1
FIG. 1
In vitro translation of the the v-cyclin/v-FLIP cassette. The bicistronic plasmids (A), with monocistronic controls and empty vector, were transcribed in vitro. RNA was analyzed on a 1% agarose-formaldehyde gel (B) and translated in reticulocyte lysate as described in Materials and Methods. (C) Labeled proteins resolved by SDS–15% PAGE. HA, hemagglutinin.
FIG. 2
FIG. 2
Mapping a region upstream of v-FLIP which allows translation. The portions of the v-cyclin/v-FLIP cassette (A) and a previously characterized IRES from EMCV (25) were cloned between the coding sequences for the subunits of heterodimeric cytokine IL-12. The bicistronic plasmids, with monocistronic controls and empty vector, were transcribed in vitro. RNA was analyzed on a 1% agarose-formaldehyde gel (B) and translated in reticulocyte lysate as described in Materials and Methods. (C) Labeled proteins resolved by SDS–12.5% PAGE.
FIG. 3
FIG. 3
Function of the v-cyclin/v-FLIP transcript in mammalian cells. 293T cells were transiently transfected with parental pcDNA 3.1(+) vector or with vectors containing the coding sequence for bicistronic v-cyclin/v-FLIP. After 48 h, half the cells were used to prepare RNA as described in Materials and Methods and half were lysed in radioimmunoprecipitation assay buffer for SDS-PAGE. (A) RNA probed with a v-FLIP probe. (B) v-cyclin and v-FLIP detected on a Western blot as described in Materials and Methods.
FIG. 4
FIG. 4
Mapping a region upstream of v-FLIP which allows translation in mammalian cells. 293T cells were transiently transfected with parental pcDNA 3.1(+) vector or with vectors containing sequences from the v-cyclin/v-FLIP-coding region of KSHV between the coding sequences for the subunits of the heterodimeric cytokine IL-12. The same plasmid containing the IRES from EMCV was used for comparison. After 48 h, cell supernatant was collected and the cells were used to prepare RNA as described in Materials and Methods. (A) RNA probed with a p40 probe. (B) Level of p40 protein in supernatant detected by ELISA, as described in Materials and Methods. p40 levels are shown as the means of triplicate determinations with standard errors of the means (SEM).
FIG. 5
FIG. 5
v-FLIP protein expression in KSHV-infected B cells. (A) Western blot of 70 μg of protein from the EBV-transformed B.45 cells and the KSHV-transformed EBV-negative PEL BC3 and BCP1 cells and protein from BC3 cells uninduced or induced for 48 h with 200 ng of tetradecanoyl phorbol acetate (TPA)/ml and separated by SDS–14% PAGE. Immunoblotting with a polyclonal anti-v-FLIP antiserum was performed as described in Materials and Methods. (B) Western blot of 70 μg of protein from EBV-negative B-cell line DG75 and the KSHV-transformed PEL BC3 cells separated by SDS–14% PAGE. Immunoblotting with monclonal anti-v-FLIP antibody 6/14 was performed as described in Materials and Methods.
FIG. 6
FIG. 6
BC3 cells (×500 magnification) stained with a second-layer antibody only (A) or monoclonal anti v-FLIP antibody 6/14, where a brown stain in the cytoplasm was detected (B) as described in Materials and Methods.
FIG. 7
FIG. 7
Synthesis of LNA-1 and v-FLIP during apoptosis of PEL cells. BC3 cells were serum starved for 20 h to induce apoptosis, which was detected by ANNEXIN-V Fluos staining (B) as described in Materials and Methods. Viable cells (107) were then pulsed for 2 h with labeled methionine and cysteine, after which LNA-1 and v-FLIP were immunoprecipitated as described in Materials and Methods and the immunoprecipitates were separated by SDS–7 or 14% PAGE, respectively (A). The relative levels of labeled protein were determined using densitometry software on a phosphorimager as described in Materials and Methods. The FLIP/LNA-1 labeled-protein ratio was normalized to 1 in cells with serum; the relative FLIP/LNA-1 labeled-protein ratio in cells without serum was 3.25. In a second experiment cells were serum starved for 24 h and the relative FLIP/LNA-1 labeled-protein ratio increased from 1 to 3.1 on serum starvation.
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