Identification and analysis of the K5 gene of Kaposi's sarcoma-associated herpesvirus

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8 (HHV-8), belongs to the gammaherpesvirus subfamily and encodes approximately 80 open reading frames (ORFs). Among them are a few candidates for immediate-early genes (e.g., K5). We developed a monoclonal antibody (MAb), 328C7, against the K5 antigen. This MAb reacted with the K5 gene product by immunoscreening of a cDNA library from BCBL-1 cells, and this result was confirmed by transfection of the K5 ORF into Cos-7 cells. After induction of lytic infection by treatment with 12-O-tetradecanoylphorbol-13-acetate, MAb 328C7 reacted with an antigen in the cytoplasm of BCBL-1 and BC-3 cells as early as after 4 h of induction. Immunoelectron microscopy showed that the K5 antigen was situated mainly in the endoplasmic reticulum but was not present on the virion or in the nucleus. Northern blotting with a K5-specific probe revealed a single transcript of 1.2 kb, while Western blotting showed the antigen to be a 36-kDa polypeptide. The 5' and 3' ends were then determined by rapid amplification of cDNA, followed by sequencing of RACE products, and a splice was revealed upstream of the K5 ORF. K5 expression was unaffected by the respective DNA and protein synthesis inhibitors phosphonoformic acid and cycloheximide plus actinomycin D, confirming its immediate-early nature. Transient-transfection assays showed that the K5 promoter was transactivated by ORF 50 (KSHV Rta), a homolog of Epstein-Barr virus Rta, but the K5 gene product exhibited no transregulation of its own promoter or those of DNA polymerase and the human immunodeficiency virus type 1 long terminal repeat. This is the first such analysis of an immediate-early gene product; determination of its specific biological function requires further investigation.

FIG. 1

Fluorescence (FITC) photomicrographs showing MAb 328C7 immunoreactivity in HHV-8-infected, uninfected, and transfected cells after selected periods of induction or after transfection. BCBL-1 and Raji cells were fixed and labeled after 12 and 24 h of exposure to TPA, respectively; pcDNA3-K5-transfected Cos-7 cells were fixed and labeled 24 h posttransfection. a, untreated BCBL-1 cells; b, TPA-treated BCBL-1 cells; c, TPA-treated Raji cells; d, TPA-treated BCBL-1 cells; e, Cos-7 cells transfected with pcDNA3-K5 also stained with Hoechst 33342.

FIG. 2

Electron micrographs demonstrating the presence of K5 antigen in BCBL-1 cells. Immunogold labeling (arrows) is detected only on membranes of endoplasmic reticulum-like structures (A and B) but not in a nucleus (N) and mitochondria (M). Immunogold labeling was performed using cryo-thin sections (gold particles, 5 nm in diameter). Original magnifications: A, ×57,000; B, ×60,000.

FIG. 3

Western blot analysis of cell lysates obtained from TPA-treated (lane 1) and untreated (lane 2) BCBL-1 cells and untreated Raji cells (lane 3) using MAb 328C7 as a probe. Polypeptides were separated by SDS–10% PAGE under reducing conditions, transferred to a polyvinylidene difluoride membrane, and reacted with MAb 328C7. Molecular mass markers are shown on the left. The arrow indicates the HHV-8-specific polypeptide recognized by MAb 328C7.

FIG. 4

RT-PCR and nested PCR of K5. Using DNase I-digested poly(A) RNA extracted from untreated and TPA-treated BCBL-1 cells, a spliced fragment encoding K5 was amplified by RT-PCR and subsequent nested PCR. (A) Schematic representation of the primers used in the assays. (B) Nested PCR results obtained with primers K5F and K5R3. Lanes: M, molecular weight markers (φX174 HaeIII digest); 1, untreated cells; 2, TPA-treated cells; 3, control PCR with HHV-8 genomic DNA.

FIG. 5

Map showing the K5 transcription pattern. (A) Schematic representation of the primer used in the primer extension assay and summary of the maps obtained using primer extension and 5′ and 3′ RACE. (B) Initiation site of the K5 transcript identified by primer extension. The size of the primer extension product is indicated by a sequencing ladder initiated with the same primer. The arrowhead indicates the position of the cDNA product, and the number indicates its position with respect to the initiation codon (ATG).

FIG. 6

Map of the K5 ORF. A putative CCAT box and the poly(A) signal are boxed and in boldface; note that they lack a distinguishable TATA box. The asterisk indicates the initiation site of the K5 transcript. The nucleotide sequence corresponding to the intron is boxed and shaded. The splice donor (GT…) and splice acceptor (…AG) sites are in boldface. The primers (K5F, K5F1, K5R1, K5R2, and K5R3) used for 3′ and 5′ RACE and RT-PCR are underlined. The arrows indicate the orientations of the primers.

FIG. 7

Fluorescence (FITC) photomicrographs showing the time course of K5 protein expression. BC-3 cells were left uninduced or induced with TPA. After 2, 4, 6, 8, and 12 h of induction, cells were labeled with MAb 328C7.

FIG. 8

(A) Northern blot analysis demonstrating the resistance of K5 transcription to the presence of inhibitors of DNA and protein synthesis (PFA and CHX, respectively). Total cellular RNA was prepared from TPA-stimulated BCBL-1 cells after 12 h of exposure to PFA and CHX, size fractionated on a 1% agarose-formaldehyde gel, transferred to nylon membrane, and probed with K5- and K9-specific antisense DNA probes. From top to bottom, the autoradiograms shown are of blots hybridized with probes for the K5, K9, and β-actin (used as a loading control) genes, respectively. (B) Immunoprecipitation of K5 protein expressed in BCBL-1 cells following metabolic labeling with PFA and CHX. For detection of HHV-8 IE and E proteins, cells were induced with TPA and incubated for 12 h with PFA at 300 μg/ml. For detection of HHV-8 IE genes, cells were induced with TPA and incubated for 4 h with CHX at 100 μg/ml and then for 6 h with AcD at 10 μg/ml. Labeling with [³⁵S]methionine was performed 1 h before cells were harvested. Lysates were prepared and immunoprecipitated with MAb 328C7. Samples were separated by SDS–10% denaturing PAGE. Molecular mass markers are shown on the left (lane 1). The position of the 36-kDa protein is indicated. Samples in lanes 2, 3, 4, 5, and 6 are TPA treated, TPA-PFA treated, untreated, CHX treated, and CHX-AcD treated, respectively.

FIG. 9

Responsiveness of the K5 gene promoter to the K5 gene and KSHV Rta. Transfected cells were harvested 24 h after transfection, and luciferase activity was assayed. Shown are the means ± standard deviations of triplicate transfections.