High expression of HHV-8-encoded ORF73 protein in spindle-shaped cells of Kaposi's sarcoma

Abstract

Human herpesvirus 8 (HHV-8) has been demonstrated previously in Kaposi's sarcoma (KS) tissues by immunohistochemistry, in situ polymerase chain reaction, and in situ hybridization. The HHV-8-encoded protein ORF73 is a 222- or 234-kd protein named latent nuclear antigen (LNA) or latency-associated nuclear antigen (LANA) that is identified in HHV-8-infected cell lines by immunofluorescence assay. In the present study, a rabbit antibody against a recombinant ORF73 protein was developed. Immunofluorescent staining of a HHV-8-infected cell line, TY-1, showed that the staining pattern of the anti-ORF73 antibody overlapped completely the LANA staining pattern obtained using KS patients' sera. Immunoblotting analysis showed that the anti-ORF73 antibody reacted specifically with 222- and 234-kd proteins that were present in TY-1 and BCBL-1 cell lysates. Immunohistochemistry using a catalyzed signal amplification system demonstrated that the anti-ORF73 antibody reacted exclusively with the majority of KS spindle-shaped cells, showing a nuclear dot-like staining pattern. Some of the ORF73 protein-positive cells also expressed CD34 and vimentin but not CD68 or factor-VIII-related antigen. These data indicate that the anti-ORF73 antibody recognizes LANA and that most KS cells are infected with HHV-8 in the latent phase. Our findings also suggest that ORF73 protein plays an important role in the pathogenesis of KS.

Figure 1.

Immunofluorescence assay using a rabbit antibody to recombinant ORF73 protein, PA1–73N, and AIDS-KS serum. Double labeling of IFA was carried out on TY-1 cells by using PA1–73N (a) and AIDS-KS serum (b). Texas-red-conjugated anti-rabbit and FITC-conjugated anti-human IgG antibodies were used as secondary antibodies. c: A dual image indicated that the staining of PA1–73N was overlapped completely on that of AIDS-KS serum.

Figure 2.

Western blot analysis of PA1–73N and AIDS-KS serum. Lane 1, BCBL-1 (HHV-8-positive, EBV-negative cell line); lane 2, TY-1 (HHV-8-positive, EBV-negative cell line); lane 3, Raji (HHV-8-negative, EBV-positive B-cell line); lane 4, Molt-4 (HHV-8-negative, EBV-negative T-cell line); lane 5, GST protein (26 kd, open arrowhead); lane 6, recombinant GST-ORF73 protein (approximately 53 kd, closed arrowhead). a: PA1–73N recognized several proteins ranging from 120 to 234 kd. These bands were detected in the cell lysates of two HHV-8-infected PEL cell lines, BCBL-1 and TY-1, but not in two HHV-8-negative cell lines, Raji and Molt-4. The recombinant GST-ORF73 was stained with PA1–73N, whereas GST protein was not stained. b: The recombinant GST-ORF73 protein blocked the reaction of anti-ORF73 rabbit antibody with the 53-kd protein and recombinant GST-ORF73 protein completely. c: AIDS-KS serum reacted with several proteins, including 120- to 234-kd proteins in BCBL-1 and TY-1 cells. AIDS-KS serum also reacted with a recombinant GST-ORF73 protein.

Figure 3.

Immunohistochemistry of ORF73 in KS. a: Hematoxylin and eosin staining of a biopsy specimen of nodular KS. b: Demonstration of the HHV-8 lytic protein. ORF59 antigen is detected in a small number of KS cells. c and d: Stained with affinity-purified rabbit antibodies to ORF73. A large proportion of KS tumor cells expressed ORF73 antigen in their nuclei. e and f: ORF73 antigen is demonstrated exclusively in KS lesion in an inguinal lymph node. Any lymphocytes do not express ORF73 antigen.