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. 2008 May;82(9):4562-72.
doi: 10.1128/JVI.02400-07. Epub 2008 Feb 27.

Identification and characterization of the product encoded by ORF69 of Kaposi's sarcoma-associated herpesvirus

R Santarelli  1 , A Farina, M Granato, R Gonnella, S Raffa, L Leone, R Bei, A Modesti, L Frati, M R Torrisi, A Faggioni
Affiliations

Identification and characterization of the product encoded by ORF69 of Kaposi's sarcoma-associated herpesvirus

R Santarelli et al. J Virol. 2008 May.

Abstract

We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.

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Figures

FIG. 1.
FIG. 1.
Amino acid sequence comparison between the putative protein encoded by KSHV ORF69 (p33) and its homologs in human herpesviruses: HSV-1 UL31, CMV UL53, and EBV BFLF2. Sequences were aligned with the MacVector 7.0 program. Identical and similar amino acid residues are highlighted by dark and light solid boxes, respectively. Numbers indicate amino acid positions.
FIG. 2.
FIG. 2.
ORF69 transcription analysis in BCBL-1 cells. (A) Kinetics of ORF69 expression upon chemical induction in BCBL-1 cell line. Cells were untreated or induced with TPA for 24, 48, or 72 h; total RNA was prepared at the times indicated, and 20 mg was analyzed by Northern blotting. As a negative control, RNA from DG75 treated with TPA for 72 h was loaded. The filter was hybridized with an oligonucleotide specific for ORF69 (RS3 probe) and subsequently with a β-actin probe. The two specific ORF69 transcripts are shown with arrowheads. (B) ORF68-ORF69 bicistronic transcript. Reverse transcription was performed on total RNA from BCBL-1 cells treated with TPA for 48 h by using oligo(dT), and PCR was carried out with primers I and II. Nucleotide sequences of primers I and II are described in Materials and Methods. Lanes: M, marker; 1, PCR without reverse transcription; 2, RT-PCR. (C) 5′-3′ RACE analysis of ORF69 transcript. Schematic drawing of the location of ORF69 in the KSHV genome. Numbers correspond to nucleotide positions, and arrows indicate the direction ORF68 and ORF69. The ORF69 transcription start is at position 116515. TATAAAAA is the putative TATA box. AATAAA is the polyadenylation recognition sequence.
FIG. 3.
FIG. 3.
Identification of p33. (A) Reactivity of H8 monoclonal antibody. Protein extracts from BL21(DE3)Lys transformed with pGEX2T, pET30a, pGEX2T-ORF69 (ORF69-GST), or pET30a-ORF69(ORF69-His) were analyzed by Western blotting after IPTG induction. (B) Expression of ORF69-encoded protein in different PEL cell lines analyzed by Western blotting. The H8 monoclonal antibody revealed a 33-kDa-specific band (p33) after KSHV lytic cycle induction with TPA in BCBL-1, BC3, and BC1. B95-8 cells were treated with TPA and butyrate. Anti-BFLF2 antibody showed EBV activation. Anti-β-actin was used to calibrate the protein loading.
FIG. 4.
FIG. 4.
p33 intracellular localization. (Top panels) Indirect immunofluorescence on BCBL-1 and BC1 cell lines stimulated with 20 ng/ml of TPA for 48 h and incubated with PP27 rabbit polyclonal antibody against p33. At least 10 optical sections (0.5 μm) per cell were obtained, and a selection is shown as a gallery. Nuclei are stained with DAPI. p33 is distributed in dots within the nucleus as well as on portions of the nuclear membrane (arrows). (Middle panels) Confocal microscopy performed for the BC3 cell line after 48 h of treatment with TPA; staining was with rabbit anti-p33 antibody and anti-K8.1A monoclonal antibody. K8.1A is present both in the cytoplasm and on the plasma membrane. Merged images do not show any p33 colocalization with K8.1A. (Bottom panels) Confocal microscopy of TRExBCBL-1-Rta cells after treatment with doxycycline and incubation with anti-p33 monoclonal antibody, together with anti-Rta polyclonal antibody. Both p33 and Rta are localized within the nucleus, as expected. Merge reveals colocalization of the two signals in a few intranuclear spots. Bars = 10 μm.
FIG. 5.
FIG. 5.
p33 is a nuclear matrix-associated protein. (A) Scheme of high-salt nuclear matrix fractionation protocol from Ben-Yehoyada et al. (2). (B) Distribution of p33 in the different fractions. Nuclear matrix fractionation was performed on the BCBL-1 cells that were untreated (−) or were induced with TPA for 48 h (+). Proteins were analyzed by SDS-PAGE and immunoblotting. The membrane was incubated with goat anti-lamin B (top panel) and mouse anti-p53 (bottom panel) as a control for the fractions obtained. H8 monoclonal antibody detects p33 predominantly in the nuclear matrix fraction (middle panel). I, II, III, and IV: fractions after high-salt purification. MW, molecular mass in kilodaltons.
FIG. 6.
FIG. 6.
p33 is not detected in HHV-8 mature virions. (A) Western blot analysis was performed to evaluate the presence of p33 in the extracellular virions. BCBL-1 cells were synchronized in S phase and treated with Na-butyrate for 72 h. Total cellular lysates from uninduced (−) BCBL-1 cells, from Na-butyrate-treated BCBL-1 cells (+), and from PEG-purified virion lysates (V) were run on SDS-PAGE gels and immunoblotted. Incubation with H8 shows that p33 is not present in extracellular virions. Anti-K8.1A monoclonal antibody was used as a control for extracellular KSHV. (B) Immunoelectron microscopy with rabbit anti-p33 of KSHV particles in induced BCBL-1. Only intracellular KSHV nucleocapsids in the perinuclear area (+), not extracellular virions (V), were immunogold labeled.
FIG. 7.
FIG. 7.
Intracellular localization of ectopically expressed p33. Human embryonic kidney 293 cells were transfected with ORF69 and, after 24 h, were stained with H8 monoclonal antibody. IFA analysis shows that p33 is confined to the nuclei of transfected cells (top panels). ORF69 cotransfection with EGFP-ORF67 in 293 cells (bottom panels). Cells were immunostained with H8 monoclonal antibody detected with sheep anti-mouse Cy3 24 h after transfection. p33 is localized at the nuclear rim in the cotransfected cells, and merged images show colocalization of the two signals. As a control, 293 cells were transfected with pEGFP empty vector (middle panels). Nuclei were stained with DAPI. Bars = 10 μm.
FIG. 8.
FIG. 8.
Indirect immunofluorescence of 293 cells transfected with EGFP-ORF69 or cotransfected with EGFP-ORF69 and BFRF1. p33 is localized diffusely within the nucleus in cells transfected with EGFP-ORF69 alone and does not colocalize with nuclear pore complexes or the endoplasmic reticulum (calreticulin) (left panels). On the contrary, the presence of BFRF1 modifies the localization of p33, which colocalizes on the nuclear membrane with BFRF1, nuclear pores, and the endoplasmic reticulum (right panels). Bars = 10 μm.
FIG. 9.
FIG. 9.
Electron microscopic analysis of the nuclear membrane structure in transfected 293 cells. (A) Cells transfected with ORF69. (B) Cells transfected with BFRF1. (C) Cells cotransfected with ORF69 and BFRF1. In cells expressing BFRF1 alone (B), large areas of nuclear membrane reduplications are evident, which cannot be observed in ORF69-expressing cells (A). In cells expressing both BFRF1 and ORF69 (C), the overall ultrastructure of the nuclear membrane appears drastically modified, showing interrupted areas of thick and irregular multilayering.
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