N. BALACHANDRAN,* ROBERTA E. AMELSE, WEI W. ZHOU, AND CHEOW K. CHANG. Department ofMicrobiology, Molecular Genetics and Immunology, The ...
JOURNAL OF VIROLOGY, June 1989, p. 2835-2840
Vol. 63, No. 6
0022-538X/89/062835-06$02.00/0 Copyright © 1989, American Society for Microbiology
Identification of Proteins Specific for Human Herpesvirus 6-Infected Human T Cells N. BALACHANDRAN,* ROBERTA E. AMELSE, WEI W. ZHOU, AND CHEOW K. CHANG Department of Microbiology, Molecular Genetics and Immunology, The University of Kansas Medical Center, Kansas City, Kansas 66103 Received 9 November 1988/Accepted 22 February 1989
Proteins specific for human herpesvirus 6 (HHV-6)-infected human T cells (HSB-2) were examined by using polyclonal rabbit antibodies and monoclonal antibodies against HHV-6-infected cells and human sera. More than 20 proteins and six glycoproteins specific for HHV-6-infected cells were identified from [35S]methionineand [3H]glucosamine-labeled total-cell extracts. Polyclonal rabbit antibodies immunoprecipitated 33 [35S]methionine-labeled HHV-6-specific polypeptides with approximate molecular weights ranging from 180,000 to 31,000. In immunoprecipitation and Western immunoblot reactions, a patient's serum also recognized more than 30 HHV-6-specific proteins and seven glycoproteins. In contrast, sera from individuals with high-titered antibodies against other human herpesviruses reacted with fewer HHV-6-infected cell proteins, and only a 1359000-Mr polypeptide was prominent. Monoclonal antibodies to HHV-6-infected cells reacted with single and multiple polypeptides specific for virus-infected cells and immunoprecipitated three distinct sets of glycoproteins, which were designated gplO5k and gp82k, gpll6k, gp64k, and gp54k, and gplO2k.
Human herpesvirus 6 (HHV-6) is a new herpesvirus, recently isolated from the peripheral blood leukocytes of six patients with lymphoproliferative disorders and acquired immunodeficiency syndrome (AIDS) (14). All six isolates were identified as belonging to the family Herpetoviridae based on the electron microscopic morphology, showing an icosahedral capsid enclosed in an envelope, 200-nm diameter of the envelope particle, and a double-standard DNA genome (11, 14). Immunological and molecular analyses have demonstrated that the new herpesvirus is distinct from other human herpesviruses (11, 14). The viral isolates replicated in freshly stimulated human umbilical cord blood or adult peripheral blood, spleen, and bone marrow mononuclear leukocytes. Since the infected cells were human B cells, the new virus was initially designated human B-lymphotrophic virus (HBLV) (8, 11, 14). Subsequently, one of the isolates (prototype strain GS) was found to infect a number of T and B lymphoblastoid cell lines and cells of megakaryocytic and neuronal origin and has now been designated HHV-6 (2). Herpesviruses were subsequently isolated from the peripheral blood lymphocytes of AIDS patients from Uganda (9), Gambia (15), the Ivory Coast (3), and Zaire (12), infants with exanthem subitum (16), and patients with chronic fatigue syndrome (1). All these isolates were identified as related to HHV-6 by their hybridization to a 9-kilobase (kb) DNA probe (pZVH14) from the prototype GS strain. Human T cells, B cells, and glial cells supported the growth of Ugandan and Gambian strains with various degrees of efficiency (9, 15). The Ivory Coast HHV-6 strain was isolated from a patient with both human T-lymphotropic virus type I and human immunodeficiency virus type 2 infections and grew in CD4+ and CD8+ peripheral blood T cells. The isolate from Zaire (Z29) infected predominantly cord blood lymphocytes of T-cell origin, and attempts to grow it in other cells have not been successful (12). Knowledge about the biology of HHV-6, including transmission and its role in human diseases other than roseola, is scanty, and emerging *
seroepidemiological studies suggest that HHV-6 infection early in life (1). The DNA of HHV-6 is estimated to contain about 170 kb (10), and this should code for at least 70 proteins. Analysis of HHV-6-specific proteins is fundamental to a rational understanding of the biology of HHV-6 and its role in human diseases and for diagnostic purposes. As an initial step towards achieving these goals, we have developed rabbit polyclonal antibodies and monoclonal antibodies against infected cells. These reagents and human sera have been used for the initial characterization of proteins specific for HHV-6-infected cells, and the results are presented here. HHV-6 prototype strain GS (HBLV) used in our studies was a gift from R. C. Gallo, D. V. Ablashi, and S. Z. Salauhuddin, National Cancer Institute. Suspension cultures of human T-cell line HSB-2 (ATCC CCL 120.1, CCRFHSB-2) grown in RPMI 1640 medium (Sigma Chemical Co.) supplemented with antibiotics and 10% heat-inactivated serum supplement (CPSR-1; Sigma) were used for virus propagation and for the preparation of antigens for various assays. For infection, uninfected HSB-2 cells were centrifuged, and the pellets were suspended in medium with 5% CPSR-1 at a concentration of 106 cells per ml. These were mixed with intact HHV-6-infected cells at an uninfected occurs
cell-infected cell ratio of 10:1 or with frozen and thawed HHV-6-infected cells. Infection resulted in a characteristic aggregation and enlargement of infected cells by 4 to 6 days post infection (PI). Electron microscopic examination of thin sections of infected cells showed large quantities of developing viral particles in the host cell nucleus as well as complete enveloped particles in cytoplasmic vacuoles and in extracellular compartments (data not shown). Infectivity titers were measured by the procedures described elsewhere (1, 2, 14). Briefly, 106 fresh HSB-2 cells were infected with dilutions of either infected-cell lysates or supernatant fluids. After 5 days, infected and uninfected cells were washed in phosphate-buffered saline (PBS), fixed on cover slides by cold acetone, and tested for the presence of viral antigens in an indirect immunofluorescence assay (IFA) with prestandardized dilutions of human serum N620 (2, 14). At 6 days
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1:10,240) and an EBV antibody titer of >1:160. In an IFA with HHV-6-infected cells, all four sera showed relatively low fluorescence even at a dilution of 1:10. None of these sera reacted with uninfected cells, and the reactivity of one of them is shown in Fig. 1, lane 7. In contrast to serum N620, all the normal sera immunoprecipitated a 135K polypeptide from HHV6-infected cells (Fig. 2, lanes 8 to 11), and longer exposure revealed faintly labeled 95K and 82K polypeptides. Under our conditions of Western blot reactions, these sera did not recognize any HHV-6-specific proteins even at a dilution of 1:50, and the 135K protein was barely detectable (data not shown). Stronger reactivity with 135K protein could be attributed to HHV-6 infection in the past or could be due to cross-reactivity with antigenic determinants of other herpesviruses. Since normal sera were limited in their reactivity with HHV-6 proteins, it can be inferred that the specific reactivity to HHV-6 detected in this serum is due to specific antibodies. The data also suggest minimal antigenic crossreactivity between HHV-6 and other human herpesviruses. Monoclonal antibodies were developed to identify individual proteins specific for virus-infected cells. Four- to 6week-old BALB/c mice were immunized with three weekly
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FIG. 2. Identification of proteins specific for HHV-6-infected cells by human sera. Uninfected HSB-2 cells and HHV-6-infected cells were labeled with [35S]methionine or with [3H]glucosamine. Reactivity of serum N620 with [35S]methionine-labeled uninfected and infected cells (lanes 1 and 2) and with [3H]glucosamine-labeled uninfected and infected cells (lanes 3 and 4). Reactivity of the serum with Western blotted total cell extracts of uninfected cells (lane 5) and HHV-6-infected cells (lane 6). Lanes 7 to 10, Reactivity of sera from individuals with high-titered antibodies against other human herpesviruses with HHV-6-infected [35S]methionine-labeled cells. Lanes 7 and 8, Serum NB with uninfected and infected cells. Lanes 9 to 11, Sera LHF, LR, and JJ with HHV-6-infected cells. Arrows indicate approximate molecular weights of proteins specific for HHV-6-infected cells.
intraperitoneal injections of 3 x 10 infected cells. Preimmune serum did not react with any proteins (Fig. 3, lanes 1 and 2), and in contrast, serum from an immunized mouse immunoprecipitated several polypeptides from uninfected 1
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and HHV-6-infected cells (Fig. 3, lanes 3 and 4). 180K, 135K, 116K, 110K, 105K, 102K, 95K, 90K, 82K, and 41K proteins were specific for HHV-6-infected cells, and this reactivity did not diminish even after absorption of the 8
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FIG. 3. Identification of proteins specific for HHV-6-infected cells by monoclonal antibodies. Immunoprecipitation was done with uninfected and HHV-6-infected HSB-2 cells labeled with [35S]methionine. Lanes 1 and 2, Normal mouse serum with uninfected and infected cells; lanes 3 and 4, immune mouse serum with uninfected and infected cells. Arrows indicate proteins specific for HHV-6-infected cells. Reactivities of monoclonal antibodies with uninfected (lanes 5, 7, 9, 12, and 13) and HHV-6-infected (lanes 6, 8, 10, 11, and 14) cells. 2D6, lanes 5 and 6; 7A2, lanes 7 and 8; 6A5H7, lanes 9 and 10; 9A5D12, lanes 11 and 12; 12B3G4, lanes 13 and 14.
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serum with uninfected cells (data not shown). Spleen from this mouse was fused with Sp2/O Ag.14 myeloma cells according to procedures described elsewhere (4, 7). Several hybridomas secreting HHV-6-specific antibodies were initially selected on the basis of their specific reactivity with HHV-6 infected cells in an enzyme immunoassay and IFA. Culture supernatants from these clones were next used in immunoprecipitation reactions with [39S]methionine-labeled uninfected and infected cells, and hybridoma clones that were immunoprecipitating HHV-6-specific peptides were cloned twice and rescreened. None of our monoclonal antibodies precipitated detectable polypeptides from uninfected cells (Fig. 3, lanes 5, 7, 9, 12, and 13). Each antibody, however, precipitated one or more [3jS5]methionine-labeled polypeptides from HHV-6infected cells (Fig. 3, lanes 6, 8, 10, 11, and 14). Distinctly different immunoprecipitation patterns were obtained by these monoclonal antibodies, indicating that antibodies had different specificities. Two polypeptides, a broadly migrating 82K polypeptide and a diffused 105K polypeptide, were immunoprecipitated by monoclonal antibody 2D6 (Fig. 3, lane 6); longer exposure of the autoradiograph also revealed several faintly labeled 116K, 72K, 38K, 36K, and 31K polypeptides. Monoclonal antibody 7A2 reacted with a prominent 102K polypeptide and a faintly labeled 74K polypeptide (Fig. 3, lane 8). Three strongly labeled 116K, 64K, and 54K polypeptides and faintly labeled 105K, 60K, and 45K polypeptides were immunoprecipitated by 6A5H7 monoclonal antibody (Fig. 3, lane 10). Monoclonal antibody 9A5D12 immunoprecipitated 110K and 41K polypeptides (Fig. 3, lane 11), while a single 135K polypeptide was recognized by antibody 12B3G4 (Fig. 3, lane 14). Crossabsorption experiments showed that the proteins recognized were antigenically distinct, and all these monoclonal antibodies were very specific in their reaction with virus-infected cells in IFA (data not shown). Since hybridoma lines secreting HHV-6-specific antibodies were cloned twice, it is unlikely that precipitation of multiple peptides was due to the polyclonal nature of the antibodies, and it is most likely due to precursor-product relationships or shared determinants and/or due to precipitation of proteins in complexes. When tested with [3H]glucosamine-labeled extracts, monoclonal antibodies immunoprecipitated several glycoproteins (Fig. 4, lanes 1 to 7), and none of them reacted with control cells (data not shown). Antibodies 2D6, 13D6, and 2D4 reacted with two broadly migrating glycoproteins, gplOSk and gp82k (Fig. 4, lanes 1, 3, and 4). In addition, these antibodies also immunoprecipitated faintly labeled glycoproteins gpll6k, gp38k, gp36k, and gp3lk (Fig. 4, arrows), which might represent precursor and/or cleavage products. Antibodies 6A5H7. 6A5G3, and 14B3 immunoprecipitated three glycoproteins, gpl16k, gp64k, and gp54k (Fig. 4, lanes 2, 5, and 6). Monoclonal antibody 7A2 immunoprecipitated gplO2k and faintly labeled glycoproteins gp72k to gplOSk (Fig. 4, lane 7). Antibodies 9A5D12 and 12B3G4 did not react with any glycoproteins from HHV-6-infected cells (data not shown). These results demonstrate the presence of three glycoproteins of different specificities in HHV-6-infected cells, and further studies of processing and synthesis of these proteins, determination of virion association, and neutralizing ability of these antibodies are currently in progress. Identification, isolation, and biochemical and functional characterization of HHV-6 proteins are fundamental to a rational analysis of the biology of HHV-6 and for diagnostic purposes. This paper provides the first report of the identi-
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