KSHV-infected Cell Lines Research Articles

ITIME.219: An Immortalized KSHV Infected Endothelial Cell Line Inducible by a KSHV-Specific Stimulus to Transition From Latency to Lytic Replication and Infectious Virus Release.

Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV-8) is the causative agent of Kaposi’s sarcoma and two B cell lymphoproliferative disorders: primary effusion lymphoma and KSHV-associated multicentric Castleman’s disease. These distinct pathologies involve different infected cell types. In Kaposi’s sarcoma, the virus is harbored in spindle-like tumor cells of endothelial origin, in contrast with the two pathologies of B cells. These distinctions highlight the importance of elucidating potential differences in the mechanisms of infection for these alternate target cell types and in the properties of virus generated from each. To date there is no available chronically KSHV-infected cell line of endothelial phenotype that can be activated by the viral lytic switch protein to transition from latency to lytic replication and production of infectious virus. To advance these efforts, we engineered a novel KSHV chronically infected derivative of TIME (telomerase immortalized endothelial) cells harboring a previously reported recombinant virus (rKSHV.219) and the viral replication and transcription activator (RTA) gene under the control of a doxycycline-inducible system. The resulting cells (designated iTIME.219) maintained latent virus as indicated by expression of constitutively expressed (eGFP) but not a lytic phase (RFP) reporter gene and can be sustained under long term selection. When exposed to either sodium butyrate or doxycycline, the cells were activated to lytic replication as evidenced by the expression of RFP and KSHV lytic genes and release of large quantities of infectious virus. The identity of the iTIME.219 cells was confirmed both phenotypically (specific antigen expression) and genetically (short tandem repeat analysis), and cell stability was maintained following repeated serial passage. These results suggest the potential utility of the iTime.219 cells in future studies of the KSHV replication in endothelial cells, properties of virus generated from this biologically relevant cell type and mechanisms underlying KSHV tropism and pathogenesis.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded circRNAs Are Expressed in Infected Tumor Tissues and Are Incorporated into Virions.

Kaposi's sarcoma-associated herpesvirus (KSHV) has recently been found to generate circular RNAs (circRNAs) from several KSHV genes, most abundantly from K10 (viral interferon regulatory factor 4 [vIRF4]), K7.3, and polyadenylated nuclear (PAN) RNA. To define expression of these circRNAs, KSHV-infected cell lines, patient tissues, and purified virions were examined. KSHV circRNA expression was universally detected in tests of six primary effusion lymphoma (PEL) cell lines but ranged from low-level expression in BC-1 cells dually infected with tightly latent KSHV and Epstein-Barr virus to abundant expression in KSHV-only BCBL-1 cells with spontaneous virus production. Generally, the PAN/K7.3 locus broadly and bidirectionally generated circRNA levels that paralleled the corresponding linear RNA levels. However, RNA corresponding to a particular KSHV circularization site (circ-vIRF4) was minimally induced, despite linear vIRF4 RNA being activated by virus induction. In situ hybridization showed abundant circ-vIRF4 in noninduced PEL cells. All three KSHV circRNAs were isolated as nuclease-protected forms from gradient-purified virions collected from BrK.219 cells infected with a KSHV molecular clone. For circ-vIRF4, the fully processed form that is exported to the cytoplasm was incorporated into virus particles but the nuclear, intron-retaining form was not. The half-life of circ-vIRF4 was twice as long as that of its linear counterpart. The KSHV circRNAs could be detected at a higher rate than their corresponding linear counterparts by in situ hybridization in archival tissues and by reverse transcription-PCR (RT-PCR) in sera stored for over 25 years. In summary, KSHV circRNAs are expressed in infection-associated diseases, can be regulated depending on virus life cycle, and are incorporated into viral particles for preformed delivery, suggesting a potential function in early infection.IMPORTANCE KSHV has recently been found to encode circRNAs. circRNAs result from back-splicing of an upstream pre-mRNA splice donor exon-intron junction to an acceptor site, generating a covalently closed circle. This study revealed that for one KSHV region, the PAN/K7.3 locus, broadly and bidirectionally generated circRNA levels parallel corresponding linear RNA levels. Another KSHV circularization site (circ-vIRF4), however, showed expression that differed from that of the corresponding linear RNA. All KSHV circRNAs are incorporated into KSHV virions and are potentially expressed as immediate early products in newly infected cells.

Viral miRNAs Alter Host Cell miRNA Profiles and Modulate Innate Immune Responses.

Prevalence of the members of herpesvirus family in oral inflammatory diseases is increasingly acknowledged suggesting their likely role as an etiological factor. However, the underlying mechanisms remain obscure. In our recent miRNA profiling of healthy and diseased human tooth pulps, elevated expression of human herpesvirus encoded viral microRNAs (v-miRs) were identified. Based on the fold induction and significance values, we selected three v-miRs namely miR-K12-3-3p [Kaposi sarcoma-associated virus (KSHV)], miR-H1 [herpes simplex virus 1 (HSV1)], and miR-UL-70-3p [human cytomegalovirus (HCMV)] to further examine their impact on host cellular functions. We examined their impact on cellular miRNA profiles of primary human oral keratinocytes (HOK). Our results show differential expression of several host miRNAs in v-miR-transfected HOK. High levels of v-miRs were detected in exosomes derived from v-miR transfected HOK as well as the KSHV-infected cell lines. We show that HOK-derived exosomes release their contents into macrophages (Mφ) and alter expression of endogenous miRNAs. Concurrent expression analysis of precursor (pre)-miRNA and mature miRNA suggest transcriptional or posttranscriptional impact of v-miRs on the cellular miRNAs. Employing bioinformatics, we predicted several pathways targeted by deregulated cellular miRNAs that include cytoskeletal organization, endocytosis, and cellular signaling. We validated three novel targets of miR-K12-3-3p and miR-H1 that are involved in endocytic and intracellular trafficking pathways. To evaluate the functional consequence of this regulation, we performed phagocytic uptake of labeled bacteria and noticed significant attenuation in miR-H1 and miR-K12-3-3p but not miR-UL70-3p transfected primary human Mφ. Multiple cytokine analysis of E. coli challenged Mφ revealed marked reduction of secreted cytokine levels with important roles in innate and adaptive immune responses suggesting a role of v-miRs in immune subversion. Our findings reveal that oral disease associated v-miRs can dysregulate functions of key host cells that shape oral mucosal immunity thus exacerbating disease severity and progression.

Viral Bcl-2 Encoded by the Kaposi's Sarcoma-Associated Herpesvirus Is Vital for Virus Reactivation.

The Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 16 (orf16) encodes a viral Bcl-2 (vBcl-2) protein which shares sequence and functional homology with the Bcl-2 family. Like its cellular homologs, vBcl-2 protects various cell types from apoptosis and can also negatively regulate autophagy. vBcl-2 is transcribed during lytic infection; however, its exact function has not been determined to date. By using bacterial artificial chromosome 16 (BAC16) clone carrying the full-length KSHV genome, we have generated recombinant KSHV mutants that fail to express vBcl-2 or express mCherry-tagged vBcl-2. We show that the vBcl-2 protein is expressed at relatively low levels during lytic induction and that a lack of vBcl-2 largely reduces the efficiency of KSHV reactivation in terms of lytic gene expression, viral DNA replication, and production of infectious particles. In contrast, the establishment of latency was not affected by the absence of vBcl-2. Our findings suggest an important role for vBcl-2 during initial phases of lytic reactivation and/or during subsequent viral propagation. Given the known functions of vBcl-2 in regulating apoptosis and autophagy, which involve its direct interaction with cellular proteins and thus require high levels of protein expression, it appears that vBcl-2 may have additional regulatory functions that do not depend on high levels of protein expression. The present study shows for the first time the expression of endogenous vBcl-2 protein in KSHV-infected cell lines and demonstrates the importance of vBcl-2 during the initial phases of lytic reactivation and/or during its subsequent propagation. It is suggested that vBcl-2 has additional regulatory functions beyond apoptosis and autophagy repression that do not depend on high levels of protein expression.

Novel monoclonal antibodies for identification of multicentric Castleman's disease; Kaposi's sarcoma-associated herpesvirus-encoded vMIP-I and vMIP-II

Recent studies have indicated that vMIP-I and vMIP-II play important roles in the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV)-related diseases due to the effects of these proteins on vascularization. We developed monoclonal antibodies against KSHV-encoded viral macrophage inflammatory protein-I (vMIP-I) and vMIP-II to study these expression profiles and reveal the pathogenesis of KSHV-related diseases. The MAbs against vMIP-I and vMIP-II reacted to KSHV-infected cell lines after lytic induction. Both vMIP-I and the vMIP-II gene products were detected 24h post-induction with 12-O-tetradecanoylphorbol-13-acetate until 60h in the cytoplasm of primary effusion lymphoma cell lines. In clinical specimens, both vMIP-I and vMIP-II gene products were detected in the tissues of patients with multicentric Castleman's disease. On the other hand, only vMIP-II was detected in a subset of Kaposi's sarcoma. We concluded that these antibodies might be powerful tools to elucidate the pathogenesis of KSHV-related diseases.

Array-Based Transcript Profiling and Limiting-Dilution Reverse Transcription-PCR Analysis Identify Additional Latent Genes in Kaposi's Sarcoma-Associated Herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is a B-lymphotropic herpesvirus strongly linked to both lymphoproliferative diseases and Kaposi's sarcoma. The viral latency program of KSHV is central to persistent infection and plays important roles in the pathogenesis of KSHV-related tumors. Up to six polypeptides and 18 microRNAs are known to be expressed in latency, but it is unclear if all major latency genes have been identified. Here, we have employed array-based transcript profiling and limiting-dilution reverse transcription-PCR (RT-PCR) methodologies to explore this issue in several KSHV-infected cell lines. Our results show that RNAs encoding the K1 protein are found at low levels in most latently infected cell lines. The gene encoding v-IL-6 is also expressed as a latent transcript in some contexts. Both genes encode powerful signaling molecules with particular relevance to B cell biology: K1 mimics signaling through the B cell receptor, and v-IL-6 promotes B cell survival. These data resolve earlier controversies about K1 and v-IL-6 expression and indicate that, in addition to core latency genes, some transcripts can be expressed in KSHV latency in a context-dependent manner.

Genome-wide identification of binding sites for Kaposi's sarcoma-associated herpesvirus lytic switch protein, RTA

Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) encoded by ORF50 is a lytic switch protein for viral reactivation from latency. The expression of RTA activates the expression of downstream viral genes, and is necessary for triggering the full viral lytic program. Using chromatin immunoprecipitation assay coupled with a KSHV whole-genome tiling microarray (ChIP-on-chip) approach, we identified a set of 19 RTA binding sites in the KSHV genome in a KSHV-infected cell line BCBL-1. These binding sites are located in the regions of promoters, introns, or exons of KSHV genes including ORF8, ORFK4.1, ORFK5, PAN, ORF16, ORF29, ORF45, ORF50, ORFK8, ORFK10.1, ORF59, ORFK12, ORF71/72, ORFK14/ORF74, and ORFK15, the two origins of lytic replication OriLyt-L and OriLyt-R, and the microRNA cluster. We confirmed these RTA binding sites by ChIP and quantitative real-time PCR. We further mapped the RTA binding site in the first intron of the ORFK15 gene, and determined that it is RTA-responsive. The ORFK15 RTA binding sequence TTCCAGGAA TTCCTGGAA consists of a palindromic structure of two tandem repeats, of which each itself is also an imperfect inverted repeat. Reporter assay and electrophoretic mobility shift assay confirmed the binding of the RTA protein to this sequence in vitro. Sequence alignment with other RTA binding sites identified the RTA consensus binding motif as TTCCAGGAT(N) 0–16TTCCTGGGA. Interestingly, most of the identified RTA binding sites contain only half or part of this RTA binding motif. These results suggest the complexity of RTA binding in vivo, and the involvement of other cellular or viral transcription factors during RTA transactivation of target genes.

Latency-Associated Nuclear Antigen (LANA) of Kaposi's Sarcoma-Associated Herpesvirus Interacts with Origin Recognition Complexes at the LANA Binding Sequence within the Terminal Repeats

Kaposi's sarcoma-associated herpesvirus (KSHV) DNA persists in latently infected cells as an episome via tethering to the host chromosomes. The latency-associated nuclear antigen (LANA) of KSHV binds to the cis-acting elements in the terminal repeat (TR) region of the genome through its carboxy terminus. Previous studies have demonstrated that LANA is important for episome maintenance and replication of the TR-containing plasmids. Here we report that LANA associates with origin recognition complexes (ORCs) when bound to its 17-bp LANA binding cognate sequence (LBS). Chromatin immunoprecipitation of multiple regions across the entire genome from two KSHV-infected cell lines, BC-3 and BCBL-1, revealed that the ORCs predominantly associated with the chromatin structure at the TR as well as two regions within the long unique region of the genome. Coimmunoprecipitation of ORCs with LANA-specific antibodies shows that ORCs can bind and form complexes with LANA in cells. This association was further supported by in vitro binding studies which showed that ORCs associate with LANA predominantly through the carboxy-terminal DNA binding region. KSHV-positive BC-3 and BCBL-1 cells arrested in G(1)/S phase showed colocalization of LANA with ORCs. Furthermore, replication of The TR-containing plasmid required both the N- and C termini of LANA in 293 and DG75 cells. Interestingly, our studies did not detect cellular ORCs associated with packaged viral DNA as an analysis of purified virions did not reveal the presence of ORCs, minichromosome maintenance proteins, or LANA.

B-Raf–dependent expression of vascular endothelial growth factor–A in Kaposi sarcoma–associated herpesvirus-infected human B cells

AbstractKaposi sarcoma–associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) is etiologically linked to Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease. Vascular endothelial growth factor–A (VEGF-A) is one of the essential factors required in KSHV pathogenesis, mainly due to its ability to mediate angiogenesis. In this report we analyzed the relationship between Raf and VEGF-A expression in KSHV-infected hematopoietic cells. All of the KSHV-infected cell lines (derived from PEL) expressed higher levels of B-Raf and VEGF-A when compared with uninfected cells. Inhibition of Raf to mitogen-induced extracellular kinase (MEK) to extracellular signal-related kinase (ERK) signaling, either by the use of MEK inhibitor (PD98059) or by siRNA specific to B-Raf, significantly lowered VEGF-A expression. In addition, B-Raf–induced VEGF-A expression was demonstrated to be sufficient to enhance tubule formation in endothelial cells. Interestingly, we did not observe mutation in the B-Raf gene of the KSHV-infected PEL cell lines. Taken together, we report for the first time the ability of Raf-associated signaling to play a role in the expression of VEGF-A in KSHV-infected hematopoietic cells.

A polypyrimidine tract facilitates the expression of Kaposi's sarcoma-associated herpesvirus vFLIP through an internal ribosome entry site.

We have identified a novel internal ribosome entry site (IRES) within a latently expressed Kaposi's sarcoma-associated herpesvirus (KSHV) gene (vCyclin) that controls the expression of a downstream open reading frame encoding an inhibitor of apoptosis (vFLIP). This IRES is the first such element to be identified in a DNA virus and may represent a novel mechanism through which this virus controls gene expression. We have used a dual luciferase reporter assay to identify important sequence elements essential for the activity of the IRES. A sequence of 32 nucleotides incorporating a polypyrimidine tract (PPT) was found to be required for the proper functioning of the IRES. We also show, using an electrophoretic mobility shift assay (EMSA), that proteins specific to a KSHV-infected cell line (BCP-1) but not a KSHV-negative cell line (HEK293) were able to form complexes with the IRES. By using an in vitro RNA binding assay, the cellular polypyrimidine tract binding protein (PTB, hnRNP-I) was found to bind to the IRES RNA. These results suggest that the interaction of PTB with the PPT may contribute to the correct functioning of the KSHV IRES in infected cells.

Latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts with human myeloid cell nuclear differentiation antigen induced by interferon alpha.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpes virus type 8 (HHV-8) is tightly linked to the development of Kaposi's sarcoma, primary effusion lymphoma (PEL) and some cases of multicentric Castleman's disease. Latency-associated nuclear antigen (LANA) is one of a limited number of KSHV genes consistently expressed in these diseases as well as in KSHV-infected cell lines derived from PEL, and has been shown to play crucial role in persistence of KSHV genomes in the infected cells. In this study, we explored the cellular factors that interact with LANA using yeast two-hybrid screening, and isolated a part of gene encoding human myeloid cell nuclear differentiation antigen (MNDA). MNDA is a hematopoietic interferon-inducible nuclear proteins with a HIN-200 family member with conserved 200-amino acid repeats. Immunoprecipitation assay revealed that LANA interacted with MNDA in a mammalian embryonic kidney cell line. MNDA transcript was undetectable in three PEL cell lines by reverse-transcription polymerase chain reaction, but it was induced by interferon alpha (IFNalpha). Moreover, LANA and MNDA were co-localized in the nuclei of MNDA-expressing PEL cells. Our results suggest that LANA interacts with MNDA in KSHV-infected cells exposed to IFNalpha. Such interaction may modulate IFN-mediated host defense activities.

Impaired CTL recognition of cells latently infected with Kaposi's sarcoma-associated herpes virus.

Kaposi's sarcoma-associated herpes virus (KSHV) is a recently identified human gamma2-herpesvirus associated with Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease. We reasoned that CTL responses may provide host defense against this virus, and consequently, KSHV may have evolved strategies to evade the CTL-mediated immune surveillance. In this study six B cell lines latently infected with KSHV were found to express reduced levels of HLA class I surface molecules compared with B cell lines transformed by the related gamma-herpesvirus EBV. KSHV-infected cells also required higher concentrations of soluble peptides to induce efficient CTL-mediated lysis than control cell lines and were unable to process and/or present intracellularly expressed Ag. Incubation of the KSHV-infected cell lines with high concentrations of soluble HLA class I binding peptides did not restore the deficient HLA class I surface expression. To assess the underlying mechanisms of these phenomena, TAP-1 and TAP-2 gene expression was analyzed. While no attenuation in TAP-2 expression was observed, TAP-1 expression was significantly reduced in all KSHV cell lines compared with that in controls. These results indicate that KSHV can modulate HLA class I-restricted Ag presentation to CTL, which may allow latently infected cells to escape CTL recognition and persist in the infected host.

Hot-spot variations of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen and application in genotyping by PCR-RFLP.

Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus-8) is aetiologically associated with Kaposi's sarcoma and several other lymphoproliferative disorders. The latent nuclear antigen (LNA) encoded by KSHV ORF73 has important functions in virus latent infection and shows molecular polymorphism. Sequence variations were identified in the internal repeat domain (IRD) of ORF73. DNA sequencing of ORF73 from one KSHV-infected cell line, PK-1, revealed that there were 558 bp (30.2%) deletions and 66 (3.6%) point mutations located mainly in repeat region 2, the glutamine-rich region of ORF73 IRD, compared with ORF73 of BC-1 KSHV. Similar sequence variations of ORF73 were also identified in two other isolates. None of the sequence variations caused any translational frame-shift in these four KSHV isolates examined, suggesting that LNA has a conservative function in virus latent infection. The frequent sequence variations in repeat region 2 of ORF73 IRD were also identified by PCR-RFLP genotyping in 26 KSHV isolates, suggesting that this region is a 'hot-spot' for genetic variations. Each Kaposi's sarcoma lesion sample contained one virus genotype with a unique RFLP pattern, indicating that in vivo KSHV infection was established with single predominate genotypes, which was further supported by the presence of invariable genotypes in multifocal lesions from individual KS patients. Four KSHV subtypes were classified based on the RFLP patterns that represent the patterns of DNA sequence variations in the ORF73 IRD. PCR-RFLP genotyping is capable of identifying LNA genetic variations and differentiating individual KSHV isolates, and thus may be useful for KSHV molecular epidemiology studies.

Differential Viral Protein Expression in Kaposi's Sarcoma-Associated Herpesvirus-Infected Diseases: Kaposi's Sarcoma, Primary Effusion Lymphoma, and Multicentric Castleman's Disease

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is linked to KS, primary effusion lymphomas (PEL), and a subset of multicentric Castleman's disease (MCD). Transcript mapping studies using PEL cell lines have allowed preliminary classification of viral gene expression into constitutive (class I) and inducible (class II/III) categories. To determine whether viral gene expression differs in vivo, we examined tissue sections of KSHV-infected disorders, using specific antibodies against proteins that are representative of the different expression classes of KSHV genes. ORF73/LANA appears to be a surrogate marker for KSHV infection because it is constitutively expressed in vitro and in vivo in all KSHV-infected cells. Expression of vIRF1, vIL6, and PF-8 proteins in the infected B cells of MCD lymph nodes reproduces the expression pattern observed in TPA-stimulated KSHV-infected B-cell lines. In contrast, the protein expression of the inducible viral genes that we tested in KS and PEL biopsies is restricted to PF-8 and vIL6, respectively. The tightly restricted expression of KSHV proteins in vivo differs from the dysregulated expression of inducible KSHV genes in vitro and suggests that viral gene expression in KSHV-infected cell lines does not accurately reflect what occurs in diseased tissues. These differences may be related to either cell-specific or immune restriction of viral replication.

Distinct Patterns of Viral Antigen Expression in Epstein–Barr Virus and Kaposi's Sarcoma-Associated Herpesvirus Coinfected Body-Cavity-Based Lymphoma Cell Lines: Potential Switches in Latent Gene Expression Due to Coinfection

Epstein–Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), are human gammaherpesviruses associated with numerous lymphomas and proliferative diseases in humans. We were interested in the protein expression patterns of specific latent and lytic proteins from the EBV genome in two body-cavity-based lymphoma cell lines, BC-1 and BC-2, which are coinfected with EBV and KSHV. BC-1 and BC-2 were analyzed using specific antibodies to latent proteins known to be essential for EBV immortalization of human primary B-lymphocytes in vitro and lytic antigens important for EBV replication and production of viral progeny. The coinfected cell lines are compared with two singly infected KSHV cell lines to determine whether antibodies against EBV-specific proteins cross-reacted against KSHV antigens. All the KSHV-infected cell lines express the KSHV-specific latency-associated nuclear antigen (LANA) with a specific pattern in the nucleus. This staining was distinct from that seen for EBNA1 in the EBV coinfected lines BC-1 and BC-2 staining the nucleus as a diffused pattern throughout the nucleus with denser staining in some regions. The coinfected cell lines all express EBNA1 and LMP1 at lower levels compared with singly infected EBV lymphoblastoid cell lines (LCLs). However, the essential latent antigens EBNA2, EBNA3A, and EBNA3C are not expressed in BC-1 and BC-2. This indicates potential regulation of EBV latent gene expression by KSHV-encoded viral or KSHV-induced cellular gene products. Additionally, lytic gene expression analysis demonstrated that BZLF1 and BMRF1 are expressed along with other early antigens (EA-D). A specific protein is detected in a singly infected KSHV cell line with cross-reactivity to antibodies that detected the EA-D complex. Moreover, in all the cell lines infected with EBV, KSHV, or EBV and KSHV, human serum with antibodies against KSHV antigens recognizes specific viral antigens ∼110 and 41–42 kDa, suggesting that human antibodies against KSHV-specific antigens can cross-react with similar EBV antigens. Therefore these data suggest that the EBV pattern of gene expression in the coinfected cell lines is a type II pattern of latency also seen in other human tumors including nasopharyngeal carcinoma and Hodgkin's lymphoma. This distinct pattern of latent and lytic gene expression in these cell lines may provide clues as to the selection for coinfection in these body cavity based lymphomas in immunocompromised hosts.

Genetics and development Paper alert

Recent studies have indicated that vMIP-I and vMIP-II play important roles in the pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV)-related diseases due to the effects of these proteins on vascularization. We developed monoclonal antibodies against KSHV-encoded viral macrophage inflammatory protein-I (vMIP-I) and vMIP-II to study these expression profiles and reveal the pathogenesis of KSHV-related diseases. The MAbs against vMIP-I and vMIP-II reacted to KSHV-infected cell lines after lytic induction. Both vMIP-I and the vMIP-II gene products were detected 24 h post-induction with 12-O-tetradecanoylphorbol-13-acetate until 60 h in the cytoplasm of primary effusion lymphoma cell lines. In clinical specimens, both vMIP-I and vMIP-II gene products were detected in the tissues of patients with multicentric Castleman's disease. On the other hand, only vMIP-II was detected in a subset of Kaposi's sarcoma. We concluded that these antibodies might be powerful tools to elucidate the pathogenesis of KSHV-related diseases.

KSHV antibodies among Americans, Italians and Ugandans with and without Kaposi's sarcoma.

A major controversy regarding Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8) is whether or not it is a ubiquitous infection of humans. Immunoassays based on KSHV- and Epstein-Barr virus (EBV)-coinfected cell lines show that most US AIDS-KS patients have specific antibodies to KSHV-related antigens. We have developed a sensitive indirect immunofluorescence assay (IFA) based on an EBV-negative, KSHV-infected cell line, BCP-1. When we used this IFA assay, KSHV-related antibodies were found in 71-88% of serum samples from US, Italian and Ugandan AIDS-KS patients, as well as all serum samples examined from HIV-seronegative KS patients. Although none of the US blood donors examined were KSHV seropositive by IFA, intermediate and high seroprevalence rates were found in Italian and Ugandan control populations. Antibody kinetics showed that more than half of the AIDS-KS patients who were examined IgG-seroconverted before KS development, and antibody levels did not decline after seroconversion. For these patients, seropositivity rates increased linearly with time, suggesting that the rate of infection was constant and that the risk of developing KS once infected with KSHV is not highly dependent on the duration of infection. These data strongly suggest that KSHV is not ubiquitous in most populations and that the virus may be under strict immunologic control in healthy KSHV-infected persons.

“In vivo et in vitro” assessment of the influence of ritodrine and oxytocin on the placental secretion of human chorionic gonadotropin and placental lactogen

Epstein–Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8 (HHV-8), are human gammaherpesviruses associated with numerous lymphomas and proliferative diseases in humans. We were interested in the protein expression patterns of specific latent and lytic proteins from the EBV genome in two body-cavity-based lymphoma cell lines, BC-1 and BC-2, which are coinfected with EBV and KSHV. BC-1 and BC-2 were analyzed using specific antibodies to latent proteins known to be essential for EBV immortalization of human primary B-lymphocytes in vitro and lytic antigens important for EBV replication and production of viral progeny. The coinfected cell lines are compared with two singly infected KSHV cell lines to determine whether antibodies against EBV-specific proteins cross-reacted against KSHV antigens. All the KSHV-infected cell lines express the KSHV-specific latency-associated nuclear antigen (LANA) with a specific pattern in the nucleus. This staining was distinct from that seen for EBNA1 in the EBV coinfected lines BC-1 and BC-2 staining the nucleus as a diffused pattern throughout the nucleus with denser staining in some regions. The coinfected cell lines all express EBNA1 and LMP1 at lower levels compared with singly infected EBV lymphoblastoid cell lines (LCLs). However, the essential latent antigens EBNA2, EBNA3A, and EBNA3C are not expressed in BC-1 and BC-2. This indicates potential regulation of EBV latent gene expression by KSHV-encoded viral or KSHV-induced cellular gene products. Additionally, lytic gene expression analysis demonstrated that BZLF1 and BMRF1 are expressed along with other early antigens (EA-D). A specific protein is detected in a singly infected KSHV cell line with cross-reactivity to antibodies that detected the EA-D complex. Moreover, in all the cell lines infected with EBV, KSHV, or EBV and KSHV, human serum with antibodies against KSHV antigens recognizes specific viral antigens ∼110 and 41–42 kDa, suggesting that human antibodies against KSHV-specific antigens can cross-react with similar EBV antigens. Therefore these data suggest that the EBV pattern of gene expression in the coinfected cell lines is a type II pattern of latency also seen in other human tumors including nasopharyngeal carcinoma and Hodgkin's lymphoma. This distinct pattern of latent and lytic gene expression in these cell lines may provide clues as to the selection for coinfection in these body cavity based lymphomas in immunocompromised hosts.