The pleiotropic effects of Kaposi's sarcoma herpesvirus

Kaposi's sarcoma herpesvirus (KSHV), or human herpesvirus 8 (HHV8), is an essential factor in the pathogenesis of Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL). Case reports suggest an occasional involvement in bone marrow hypoplasia and haemophagocytic syndrome, but other disease associations are unconfirmed or controversial. KSHV-associated disease is of particular importance in immunosuppressed individuals, in particular in patients with HIV infection and transplant recipients. KSHV establishes a latent infection in the majority of infected cells in KS, MCD, and PEL, but lytic replication occurs in a small fraction of infected cells. Viral proteins expressed during both the latent and the lytic phase of the viral life cycle contribute to the pathogenesis of KSHV-associated diseases.     

Expression of human herpesvirus 8 (HHV-8)-encoded immediate early protein, open reading frame 50, in HHV-8-associated diseases.

OBJECTIVES: To investigate the expression of the open reading frame (ORF) 50 protein, a human herpesvirus 8 (HHV-8)-encoded immediate early protein, in HHV-8-associated diseases. STUDY DESIGN/METHODS: We developed a rabbit anti-ORF50 protein polyclonal antibody, and investigated the ORF50 protein expression by immunohistochemistry using primary effusion lymphoma (PEL) cell lines and tissue sections of Kaposi's sarcoma (KS), HHV-8-associated solid lymphoma, and multicentric Castleman's disease (MCD). RESULTS: Western blot analysis revealed that this antibody reacted with a 110-kd protein in the lysate of phorbolester-stimulated HHV-8-infected PEL cell lines. Immunohistochemistry revealed that a very small population of cells expressed the ORF50 protein in KS and HHV-8-associated solid lymphoma cells, and almost all these cells expressed HHV-8-encoded latency-associated nuclear antigen. The ORF50 protein expression was also rare in the cells of PEL cell lines, and the staining pattern was diffuse or dot-like in the nuclei, overlapping partially with that of promyelocytic leukemia protein. In MCD, however, the ORF50 protein was expressed in some cells of the mantle zone of the lymphoid follicle. CONCLUSIONS: The ORF50 protein expression in vivo was considerably rare in KS, HHV-8-associated solid lymphoma, and PEL, but was more frequent in MCD. Rare expression of this transactivator protein in HHV-8-associated malignancies causes low expression levels of other lytic proteins and may play a role in the maintenance of the latent infection. This is the first report describing the expression of an immediate early protein of HHV-8 in cases of HHV-8-associated diseases.     

Identification of a major latent nuclear antigen, LNA-1, in the human herpesvirus 8 genome

OBJECTIVES: Human herpesvirus 8 (HHV-8) is strongly associated with all forms of Kaposi's sarcoma (KS) and with primary effusion lymphomas (PEL). KS patients' sera are immunoreactive against discrete nuclear localizing antigens in PEL cell lines. This study sought to identify and characterize these nuclear localizing proteins. STUDY DESIGN/METHODS: KS patients' sera were used to screen a cDNA expression library derived from a PEL cell line (BCP-1) latently infected with HHV-8. RESULTS: An HHV-8-specific cDNA clone was isolated. It encoded one partial and two complete open reading frames (ORFs): ORF 73, ORF 72 (v-cyclin), and K13, respectively. The immunodominant epitope was mapped to the C-terminal domain of ORF 73. Analysis with the KS patients' sera of HEK 293 cells transfected with a clone encompassing the complete coding region of ORF 73, ORF 72, and K13 gave a nuclear immunofluorescence pattern similar to that observed in BCP-1 cells. Western blot analysis with KS patients' sera of transfected HEK 293 cells revealed an immunoreactive protein of 220 to 230 kD that was similar to that observed previously in PEL cell lines. After induction of lytic replication of HHV-8 in BCP-1 cells with n-butyrate, we observed a major reduction in the expression of an ORF 73-specific 6.6-kb mRNA, indicating that this region is under latent control. CONCLUSIONS: These data identify a region of HHV-8 encoding for a major immunoreactive latent nuclear antigen (LNA-1), analogous to the Epstein-Barr virus latent nuclear antigens.     

Comparison of serologic assays for detection of antibodies against human herpesvirus 8

Improvement of serologic assays for detection of antibodies against human herpesvirus 8 (HHV-8) is critical to better understand its epidemiology and biology. We produced the HHV-8 latent (ORF73) and lytic (ORF65, K8.1, and glycoprotein B) antigens in the Semliki Forest virus system and evaluated their performance in immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). These assays were compared with other latent antigen-based assays, including an IFA based on primary effusion lymphoma (PEL) cells and an ELISA based on bacterially expressed ORF73 antigen, as well as with other lytic antigen-based assays, including an IFA based on induced PEL cells, a commercial ELISA based on purified virions, and ELISAs based on K8.1- and ORF65-derived oligopeptides. We used a panel of 180 serum specimens obtained from three groups expected to have high, intermediate, and low HHV-8 prevalences. Using three different evaluation methods, we found that (i) the performances of the lytic antigen-based ELISAs were almost equivalent, (ii) the lytic antigen-based assays were more sensitive than the latent antigen-based assays, and (iii) in general, IFAs were more sensitive than ELISAs based on the same open reading frame. We also found that serum specimens from healthy individuals contained antibodies cross-reactive with HHV-8 glycoprotein B that can potentially cause false-positive reactions in lytic PEL-based IFAs. Although this is not a substantial problem in most epidemiologic studies, it may confound the interpretation of data in studies that require high assay specificity. Because the K8.1-based IFA provides sensitivity similar to that of lytic PEL-based IFAs and improved specificity, it can be a useful alternative to the PEL-based IFAs.     

Comparison of Serologic Assays for Detection of Antibodies against Human Herpesvirus 8

Improvement of serologic assays for detection of antibodies against human herpesvirus 8 (HHV-8) is critical to better understand its epidemiology and biology. We produced the HHV-8 latent (ORF73) and lytic (ORF65, K8.1, and glycoprotein B) antigens in the Semliki Forest virus system and evaluated their performance in immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). These assays were compared with other latent antigen-based assays, including an IFA based on primary effusion lymphoma (PEL) cells and an ELISA based on bacterially expressed ORF73 antigen, as well as with other lytic antigen-based assays, including an IFA based on induced PEL cells, a commercial ELISA based on purified virions, and ELISAs based on K8.1- and ORF65-derived oligopeptides. We used a panel of 180 serum specimens obtained from three groups expected to have high, intermediate, and low HHV-8 prevalences. Using three different evaluation methods, we found that (i) the performances of the lytic antigen-based ELISAs were almost equivalent, (ii) the lytic antigen-based assays were more sensitive than the latent antigen-based assays, and (iii) in general, IFAs were more sensitive than ELISAs based on the same open reading frame. We also found that serum specimens from healthy individuals contained antibodies cross-reactive with HHV-8 glycoprotein B that can potentially cause false-positive reactions in lytic PEL-based IFAs. Although this is not a substantial problem in most epidemiologic studies, it may confound the interpretation of data in studies that require high assay specificity. Because the K8.1-based IFA provides sensitivity similar to that of lytic PEL-based IFAs and improved specificity, it can be a useful alternative to the PEL-based IFAs.     

Comparison of human sera reactivities in immunoblots with recombinant human herpesvirus (HHV)-8 proteins associated with the latent (ORF73) and lytic (ORFs 65, K8.1A, and K8.1B) replicative cycles and in immunofluorescence assays with HHV-8-infected BCBL-1 cells

The development of reliable, sensitive, and specific serological methods for the detection of human herpesvirus-8 (HHV-8) antibodies is critical for a thorough understanding of HHV-8 prevalence and pathogenesis. To evaluate the potential usefulness of HHV-8 proteins in measuring the responses against both latent and lytic antigens, we selected 1 latent [open reading frame (ORF) 73] antigen and 3 HHV-8 lytic antigens (ORFs 65, K8.1A, and K8.1B) previously identified as immunogenic [Virology (1998) 243, 208-217]. Full-length genomic ORF 73 and full-length ORFs 65, K8.1A, and K8.1B from the cDNA clones were cloned, expressed in bacterial and baculovirus-insect cell expression systems, and purified as GST fusion proteins. These recombinant proteins were used in Western blot reactions to test sera from 104 human immunodeficiency virus (HIV)+/Kaposi's sarcoma (KS)+ homosexual men, 77 HIV+/KS- homosexual men, and 84 age-matched HIV-/KS- men. These sera were also tested in immunofluorescence assays (IFAs) with uninduced and 12-O-tetradecanoylphorbol-13-acetate-induced B cell lymphoma-1 cells to detect antibodies against latency-associated nuclear antigens (LANA) and antibodies against lytic antigens (cytoplasmic fluorescence). These sera exhibited differential reactivities reflecting different titers of antibodies against HHV-8 proteins, and variable reactivities were seen more commonly with the sera from HIV-/KS- adult men. In the Western blot assay, 89% (93 of 104) of HIV+/KS + sera, 60% (46 of 77) of HIV+/KS- sera, and 7% (6 of 84) HIV+/KS- sera were reactive with both latent and lytic recombinant antigens. Western blot reactions with ORF 73 protein were more sensitive than LANA-IFA results. The lytic IFA and lytic Western blot (ORFs 65 and K8.1A) assays were more sensitive than the ORF 73 Western blots and LANA-IFA. With an exception of 2 sera from the HIV-/KS- group, all sera positive for lytic IFA antibodies and ORF 65 and K8.1A antibodies were also positive for latent antibodies. With few exceptions, sera positive for ORF 65 antibodies were also positive for K8.1A antibodies, and sera recognized the K8.1A protein more often than the K8.1B protein. There is a high degree of concordance between IFA and Western blot reactions, suggesting that this panel of HHV-8 recombinant proteins could detect a majority of the HHV-8-seropositive individuals. These results suggest that IFA followed by confirmation with the Western blot reactions with a panel of latent and lytic immunogenic antigens would provide a reliable, sensitive, and specific method for the detection of HHV-8 antibodies.     

Generation of a doxycycline-inducible KSHV producer cell line of endothelial origin: maintenance of tight latency with efficient reactivation upon induction

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS) and at least two B cell lymphoproliferative diseases: primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). B cells derived from PEL are latently infected, and can be induced to lytic replication by treatment with chemical agents like TPA or butyrate, which have pleiotropic effects on host cell signaling and chromatin structure. Most of these lines also display moderate levels of spontaneous lytic induction, which complicates analysis of latency. Here we describe the creation of latently infected cell lines derived from SLK endothelial cells that (i) display tight control of KSHV latency, with little spontaneous reactivation and (ii) are efficiently inducible by doxycycline, avoiding the need for pleiotropic inducing agents. These cells produce substantial quantities of infectious KSHV, and should be useful for studies of the latent-lytic switch and the impact of lytic replication on host cell biology.     

Human herpesvirus 8 (HHV8) sequence variations in HHV8 related tumours in Okinawa, a subtropical island in southern Japan

BACKGROUND: Although rare in mainland Japan, classic Kaposi's sarcoma (KS) is frequently reported in Okinawa, a subtropical island in southern Japan. Human herpesvirus 8 (HHV8) has been identified in the tumours and geographical differences occur. Aim: To sequence HHV8 in classic and AIDS associated KS in Okinawa. MATERIALS/METHODS: Eight classic KS cases, one AIDS associated KS, five granuloma pyogenicum cases, two inflammatory pseudotumours, two Castleman's disease cases, one angiosarcoma, and one primary effusion lymphoma (PEL) were studied. As a control, HHV8 positive cultured PEL cells (TY-1) were used. The presence of HHV8 sequences was evaluated by PCR and in situ hybridisation. PCR products were sequenced. RESULTS: There were no histological differences among KS resulting from the different virus genotypes. HHV8 was detected in all cases of KS, in one PEL, and one granuloma pyogenicum. Eight classic KS cases and one granuloma pyogenicum were infected with HHV8 genotype II/C (K1 region) or subtype C (ORF26 region), which had a five amino acid deletion at K1 VR2 region. An AIDS associated KS and a PEL were infected with type I/A virus. CONCLUSION: In Okinawa, classic KS cases and one granuloma pyogenicum case were infected with HHV8 genotype II/C, also classified as subtype C. AIDS associated KS and PEL were infected with a different HHV8 (genotype I/A), similar to that found in the USA. In Okinawa, HHV8 infection is more than four times higher than in mainland Japan, resulting in many cases of KS because of HHV8 genotype II/C infection.     

New immunofluorescence assays for detection of Human herpesvirus 8-specific antibodies

Several assays have been developed for detection of immunoglobulin G antibodies to Human herpesvirus 8 (HHV-8), including immunofluorescence assays (IFAs) and enzyme-linked immunosorbent assays (ELISAs). However, the specificity and sensitivity of these assays are not completely defined due to the lack of a "gold standard." Although IFAs based on primary effusion lymphoma (PEL) cell lines are used widely, the assays can be confounded by nonspecific reactions against cellular components and potential cross-reaction with antibodies against other herpesviruses. To provide more reliable IFAs, we established recombinant Semliki Forest viruses (rSFVs) expressing the HHV-8-specific proteins ORF73 and K8.1 and used BHK-21 cells infected with these rSFVs for IFA (ORF73-IFA and K8.1-IFA). Expression of the HHV-8-specific proteins at very high levels by the rSFV system allowed easy scoring for IFA and thereby increased specificity. The rSFV system also allowed detection of antibodies against glycosylation-dependent epitopes of K8.1. Titers measured by rSFV-based IFAs and PEL-based IFAs correlated well (correlation coefficients of >0.9), and concordances of seroreactivities between rSFV-based and PEL-based IFAs were >97% (kappa > 0.93). K8.1-IFA was more sensitive than either ORF73-IFA or peptide ELISAs. Using PEL-based lytic IFA as a reference assay, the sensitivity and specificity of K8.1-IFA were estimated to be 94 and 100%, respectively. HHV-8 prevalences determined by K8.1-IFA among the human immunodeficiency virus (HIV)-positive (HIV(+)) Kaposi's sarcoma (KS) patients, HIV(+) KS(-) patients, and healthy controls were 100, 65, and 6.7%, respectively, which were consistent with prior reports. Therefore, our rSFV-based IFAs may provide a specific and sensitive method for use in epidemiology studies. In addition, they will provide a basis for further development of diagnostic tests for HHV-8 infection.     

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8): key aspects of epidemiology and pathogenesis

The search for a transmissible infectious agent as the cause of Kaposi's sarcoma lead to the discovery in 1994 of Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus type 8 (HHV8). KSHV is the only human gamma2 herpesvirus (rhadinovirus) known so far, and is also associated with two other AIDS-related lymphoproliferative disorders: primary effusion lymphoma (PEL) and the plasma-cell variant of multicentric Castleman's disease (MCD). This review addresses key aspects of KSHV epidemiology, life cycle and pathogenesis, including the role played by key latent and lytic KSHV genes.     

Distinct expression of Kaposi's sarcoma-associated herpesvirus-encoded proteins in Kaposi's sarcoma and multicentric Castleman's disease

The expression of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8)-encoded proteins is herein demonstrated in Kaposi's sarcoma (KS) and multicentric Castleman's disease (MCD) in a single lymph node derived from a patient with acquired immunodeficiency syndrome. Immunohistochemistry revealed that both lytic and latent KSHV proteins were expressed in cells of the MCD lesion. KSHV-encoded viral interleukin-6 was also detected in follicular dendritic cells of the germinal center. Cytoplasmic localization of open reading frame 59 protein and latency-associated nuclear antigen suggested KSHV activation in the MCD lesion. Moreover, a high copy number of KSHV was detected in the blood. Clinically, pegylated-liposomal doxorubicin induced regression of not only KS, but also lymphadenopathy of the MCD lesion with a decrease in KSHV load and human interleukin-6 in the blood. To the best of the authors' knowledge this is the first case demonstrating differential expression of virus proteins in two KSHV-associated diseases, KS and MCD, in the same section. The case confirms lytic KSHV infection in MCD, and suggests that clinical symptoms of MCD might be closely linked with KSHV activation.     

Comparison of the immunoglobulin-G-specific seroreactivity of different recombinant antigens of the human herpesvirus 8.

The open reading frames ORF 52, ORF 65, K12, and K8.1 of the human herpesvirus 8 (HHV8) were expressed as glutathione-S-transferase (GST) fusion proteins and analysed by Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). The open reading frame (ORF) 65 and K8.1 antigens gave the highest reactivity (71%) in sera from HIV-dependent Kaposi's sarcoma (KS) patients. Therefore both antigens appear to be essential for HHV8 diagnostics, whereas ORF K12 and ORF 52 were of minor importance. Using polymerase chain reaction (PCR) out of the peripheral blood of these KS patients, 48% were detected as positive. By testing an N-terminal-deleted construct (amino acid 80-171) of ORF 65, we could show that the N-terminal region of this protein is essential to mediate full immunogenic reactivity. By analysing different deletion mutants of ORF K8.1, the major epitope was found to be located between aa 29 and 101. The prevalence of antibodies directed against the different antigens was determined for healthy blood donors to be 3-6%. The different antibody patterns obtained in HIV-patients with and without KS support the hypothesis that different antibody profiles develop during the course of KS.     

Morphogenesis of HHV8 in primary human dermal microvascular endothelium and primary effusion lymphomas

This study elucidates the morphology of HHV8 replication in human dermal endothelial cells and primary effusion lymphomas (PEL) and compares it to that seen in Kaposi sarcoma. Primary human dermal microvascular endothelial cells (DMVEC) exposed to the cell-filtered supernatant of the PEL JSC1 and PEL cell lines (KS-1, BCBL-1, BC-1, BC-3) were cultured in the presence or absence of 12- O -tetradecanoyl-phorbol-13-acetate (TPA) or butyrate. Cells were fixed in neutral-buffered glutaraldehyde, gelled in cooled agar, and processed for TEM. There was a quantitative, but not a qualitative difference in viral expression associated with no treatment or exposureto TPA orbutyrate of HHV8 in DMVEC and PEL. Two types of viral-induced intranuclear inclusions (INI) were visible at the light and ultrastructural levels. The more common INI had lighter staining material filling the nucleus, except for a rim of dense chromatin, and could be seen even beforeviral nucleocapsids (NC) werevisible. The second type of INI resembled a target formed by condensation of electron-dense material surrounded by a lighter halo and marginated heterochromatin and containing NC. Collections of coalescing electron-dense granules resembling starbursts were often present in nuclei containing either type of INI. Next to appear in productively infected cells were mature enveloped particles that formed mostly by the budding of NC into cytoplasmic vacuoles. Mature particles were also seen free on the plasma membrane. Tufts of electron-dense intermediate filaments were associated with maturing particles. Mature virions lacked an electron-dense tegument. Viral production was ultimately associated with cell lysis. It appears that HHV8 propagate in DMVEC, with and without stimulation, and have a similar morphogenesis to that seen in PEL cell lines and Kaposi sarcoma lesions. Several unique features characterize cells productively infected by HHV8.     

Lytic growth of human herpesvirus 8: morphological aspects

The human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus, is a gamma herpesvirus associated with AIDS-related body cavity-based lymphomas (BCBL), also called primary effusion lymphomas (PEL). These are a rare form of non-Hodgkin lymphomas in which HHV-8 is present, often associated with Epstein-Barr virus (EBV) infection. HHV-8 is also present in a latent state or in a state of low-level persistence in different primary effusion lymphoma-derived cell lines, such BCBL-1 cells, that lack EBV infection. This cell line was induced to produce mature virions by treatment with 12- O -tetradecanoyl phorbol-13-acetate (TPA) and the characteristic ultrastructural features of HHV-8 lytic replication were identified and compared to those of the other members of Herpesviridae family.     

Morphogenesis of HHV8 in Primary Human Dermal Microvascular Endothelium and Primary Effusion Lymphomas

This study elucidates the morphology of HHV8 replication in human dermal endothelial cells and primary effusion lymphomas (PEL) and compares it to that seen in Kaposi sarcoma. Primary human dermal microvascular endothelial cells (DMVEC) exposed to the cell-filtered supernatant of the PEL JSC1 and PEL cell lines (KS-1, BCBL-1, BC-1, BC-3) were cultured in the presence or absence of 12- O -tetradecanoyl-phorbol-13-acetate (TPA) or butyrate. Cells were fixed in neutral-buffered glutaraldehyde, gelled in cooled agar, and processed for TEM. There was a quantitative, but not a qualitative difference in viral expression associated with no treatment or exposureto TPA orbutyrate of HHV8 in DMVEC and PEL. Two types of viral-induced intranuclear inclusions (INI) were visible at the light and ultrastructural levels. The more common INI had lighter staining material filling the nucleus, except for a rim of dense chromatin, and could be seen even beforeviral nucleocapsids (NC) werevisible. The second type of INI resembled a target formed by condensation of electron-dense material surrounded by a lighter halo and marginated heterochromatin and containing NC. Collections of coalescing electron-dense granules resembling starbursts were often present in nuclei containing either type of INI. Next to appear in productively infected cells were mature enveloped particles that formed mostly by the budding of NC into cytoplasmic vacuoles. Mature particles were also seen free on the plasma membrane. Tufts of electron-dense intermediate filaments were associated with maturing particles. Mature virions lacked an electron-dense tegument. Viral production was ultimately associated with cell lysis. It appears that HHV8 propagate in DMVEC, with and without stimulation, and have a similar morphogenesis to that seen in PEL cell lines and Kaposi sarcoma lesions. Several unique features characterize cells productively infected by HHV8.