Kaposi's Sarcoma-Associated Herpesvirus ORF7 Is Essential for Virus Production

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman disease. Although capsid formation and maturation in the alpha-herpesvirus herpes simplex virus 1 are well understood, these processes in KSHV remain unknown. The KSHV ORF7, encoding the viral terminase (DNA cleavage and packaging protein), is thought to contribute to capsid formation; however, functional information is lacking. Here, we investigated the role of ORF7 during KSHV lytic replication by generating two types of ORF7 knock-out (KO) mutants (frameshift-induced and stop codon-induced ORF7 deficiency), KSHV BAC16, and its revertants. The results revealed that both ORF7-KO KSHVs showed significantly reduced viral production but there was no effect on lytic gene expression and viral genome replication. Complementation assays showed virus production from cells harboring ORF7-KO KSHV could be recovered by ORF7 overexpression. Additionally, exogenously expressed ORF7 partially induced nuclear relocalization of the other terminase components, ORF29 and ORF67.5. ORF7 interacted with both ORF29 and ORF67.5, whereas ORF29 and ORF67.5 failed to interact with each other, suggesting that ORF7 functions as a hub molecule in the KSHV terminase complex for interactions between ORF29 and ORF67.5. These findings indicate that ORF7 plays a key role in viral replication, as a component of terminase.

Keywords: Kaposi’s sarcoma-associated herpesvirus; ORF7; capsid formation; herpesvirus; lytic replication; terminase.

Figure 1

Construction of the frameshift-induced ORF7 and the stop codon-induced ORF7 KSHV BAC16 and their revertants. (a) Schematic illustration of the KSHV genome, including the ORF7 coding region. Two different types of ORF7-KO mutations (i.e., a frameshift using a one-base deletion and an insertion of three stop codons) were generated in the ORF7 coding region (nt6609–nt8696; accession number: GQ994935) of wild-type (WT)-KSHV BAC16 clones. The frameshift and insertion mutants were designated FS-ΔORF7-BAC16 and ST-ΔORF7-BAC16, respectively. Additionally, revertant clones, FS Rev-BAC16 and ST Rev-BAC16, were generated from FS-ΔORF7-BAC16 and ST-ΔORF7-BAC16, respectively, by replacement of the mutation site with the original DNA sequence. (b) The neighboring DNA sequence of the mutation sites in FS-ΔORF7-BAC16, ST-ΔORF7-BAC16, and their revertant BAC16 clones. (c) An image of the gel from agarose gel electrophoresis of the recombinant KSHV BAC16 digested with BglII enzyme. The asterisks indicate insertion or deletion of a kanamycin-resistance cassette in each BAC clone. Original images of the blotting are shown in Supplementary Figure S1.

Figure 2

The effects of ORF7 deficiency on KSHV lytic replication. (a–c) Extracellular virus production and intracellular viral DNA replication and lytic gene expression in iSLK cells harboring WT-KSHV-BAC16, frameshift-induced ORF7-KSHV-BAC16, and revertant KSHV-BAC16. The iSLK-WT, iSLK-FS ΔORF7, and iSLK-FS Rev cell lines were cultured for 72 hours with medium containing Dox and NaB to induce lytic replication. (a) KSHV genome copies of packaged viral particles in culture supernatants were measured using real-time PCR. (b) The quantities of intracellular viral DNA of Dox- and NaB-treated iSLK cells were measured using real-time PCR and normalized by the total amount of DNA. (c) The quantities of mRNA expression levels of viral lytic genes: IE, ORF16 (vBcl-2); E, ORF46 (uracil DNA glycosylase) and ORF47 (glycoprotein L); L, K8.1 (glycoprotein) in lytic-induced iSLK-WT, iSLK-FS ΔORF7, and iSLK-FS Rev cell lines. Each iSLK cell line was treated with or without 8 μg/mL Dox and 1.5 mM NaB, and cells were harvested at 72 h post-treatment. Total RNA purified from cells was subjected to RT real-time PCR. Evaluated lytic genes were normalized by GAPDH mRNA. The values obtained from Dox- and NaB-untreated iSLK-WT cells were defined as 1.0. (d–f) Extracellular virus production and intracellular viral DNA replication in iSLK cells harboring WT-KSHV-BAC16, stop codon-induced ORF7-KSHV-BAC16, and revertant KSHV-BAC16. The iSLK-WT, iSLK-ST ΔORF7, and iSLK-ST Rev cell lines were cultured with Dox and NaB for 72 h. (d) KSHV genome copies of viral particles in culture supernatants were measured using real-time PCR. (e) The quantities of intracellular viral DNA of Dox- and NaB-treated iSLK cells were normalized by the total amount of DNA. (f) The quantities of mRNA expression levels of viral lytic genes of Dox- and NaB-treated iSLK cells were measured using RT real-time PCR and normalized by GAPDH mRNA. The values obtained from Dox- and NaB-untreated iSLK-WT cells were defined as 1.0.

Figure 3

Virus production in iSLK cells harboring ORF7-deficient KSHV was recovered by exogenous ORF7 expression. Rescue of virus production in iSLK-FS ΔORF7 cells (a) and iSLK-ST ΔORF7 cells (b) by exogenous ORF7 expression. iSLK-FS ΔORF7 or iSLK-ST ΔORF7 cells were transiently transfected with ORF7-3xFLAG plasmid (or empty plasmid) and simultaneously cultured with medium containing Dox and NaB for 72 h. At 72 h after treatment and transfection, levels of KSHV genome copies of KSHV virions in culture supernatants were determined using real-time PCR. Exogenous ORF7 expression in ORF7-3xFLAG-transfected and Dox-induced iSLK cells was confirmed by Western blotting using anti-FLAG antibody. Original images of the Western blotting are shown in Supplementary Figure S2. (c,d) Evaluation of infectious virion in culture supernatants of ORF7-3xFLAG-transfected iSLK-FS ΔORF7 cells (c) and iSLK-ST ΔORF7 cells (d). At 72 h after Dox treatment and transfection, collected culture supernatants were inoculated with HeLa and HEK293T cells for infection. At 72 h post-infection, GFP-positive cells were counted by fluorescence microscopy. (a–d) “empty” and “ORF7-3F” mean the empty plasmid and ORF7-3xFLAG plasmid, respectively.

Figure 4

ORF7 induces nuclear relocalization of ORF29 and ORF67.5. HeLa cells were transfected with ORF7-3xFLAG (a), 2xS-ORF29 (b), 2xS-ORF67.5 (d), and 2xS-ORF17 (g) plasmids and stained with anti-HSP90beta (cyan) and either anti-FLAG (green) or anti-S tag (red) antibodies. (a–g) DNA was visualized with DAPI staining (white). Immunofluorescent images were obtained with an inverted confocal microscope. For colocalization with ORF7, ORF7-3xFLAG plasmids were cotransfected with 2xS-ORF29 (c) or 2xS-ORF67.5 (e) plasmids into HeLa cells. The cells were stained with anti-FLAG (green), anti-S tag (red), and anti-HSP90beta (cyan) antibodies. For colocalization of ORF29 and OR67.5 (f), 6xmyc-ORF29 was cotransfected with 2xS-ORF67.5, and HeLa cells were stained with anti-myc (green), anti-S tag (red), and anti-HSP90beta (cyan) antibodies.

Figure 5

ORF7 functions as a hub molecule for interactions between ORF29 and ORF67.5. (a) ORF7 interacted with ORF29 and ORF67.5. HEK293T cells were cotransfected with plasmids expressing ORF7-3xFLAG and either 2xS-ORF29 or 2xS-ORF67.5. Cell lysates prepared at 18 h post-transfection were subjected to a pulldown assay (Pd) using S-protein beads. The precipitated 2xS-ORF29 or 2xS-ORF67.5 was analyzed by immunoblotting (IB) using anti-FLAG antibody to detect ORF7-3xFLAG. (b) ORF29 interacted with ORF7 but not ORF67.5. Cells were cotransfected with 5xHA-ORF29 and either ORF7-2xS or 2xS-ORF67.5 plasmids. The precipitated ORF7-2xS or 2xS-ORF67.5 was analyzed by IB using anti-HA antibody to detect 5xHA-ORF29. (c) ORF67.5 interacted with ORF7 but not ORF29. Cells were cotransfected with 5xHA-ORF67.5 and either ORF7-2xS or 2xS-ORF29 plasmids. The precipitated ORF7-2xS or 2xS-ORF29 was analyzed by IB using anti-HA antibody to detect 5xHA-ORF67.5. (a–c) “Input” means total cell lysates, and 2% cell lysates used for pulldown were applied for SDS-PAGE. 2xS-LANA-C plasmid (KSHV ORF73/LANA 954-1162 C-terminal end) was used for the negative control for ORF7 binding. Original images of blotting are shown in Supplementary Figures S3 to S5.