A panel of KSHV mutants in the polycistronic kaposin locus for precise analysis of individual protein products

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the cause of several human cancers including the endothelial cell (EC) malignancy, Kaposi’s sarcoma. Unique KSHV genes absent from other human herpesvirus genomes, the “K-genes”, are important for KSHV replication and pathogenesis. Among these, the kaposin transcript is highly expressed in all phases of infection, but its complex polycistronic nature has hindered functional analysis to date. At least three proteins are produced from the kaposin transcript: Kaposin A (KapA), B (KapB), and C (KapC). To determine the relative contributions of kaposin proteins during KSHV infection, we created a collection of mutant viruses unable to produce kaposin proteins individually or in combination. In previous work, we showed KapB alone recapitulated the elevated proinflammatory cytokine transcripts associated with KS via the disassembly of RNA granules called processing bodies (PBs). Using the new ΔKapB virus, we showed that KapB was necessary for this effect during latent KSHV infection. Moreover, we observed that despite the ability of all kaposin-deficient latent iSLK cell lines to produce virions, all displayed low viral episome copy number, a defect that became more pronounced after primary infection of naïve ECs. For ΔKapB, provision of KapB in trans failed to complement the defect, suggesting a requirement for the kaposin locus in cis . These findings demonstrate that our panel of kaposin-deficient viruses enables precise analysis of the respective contributions of individual kaposin proteins to KSHV replication. Moreover, our mutagenesis approach serves as a guide for the functional analysis of other complex multicistronic viral loci. Importance Kaposi’s sarcoma-associated herpesvirus (KSHV) expresses high levels of the kaposin transcript during both latent and lytic phases of replication. Due to its repetitive, GC-rich nature and polycistronic coding capacity, until now no reagents existed to permit a methodical analysis of the role of individual kaposin proteins in KSHV replication. We report the creation of a panel of recombinant viruses and matched producer cell lines that delete kaposin proteins individually or in combination. We demonstrate the utility of this panel by confirming the requirement of one kaposin translation product to a key KSHV latency phenotype. This study describes a new panel of molecular tools for the KSHV field to enable precise analysis of the roles of individual kaposin proteins during KSHV infection.

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Efficient Infection by a Recombinant Kaposi's Sarcoma-Associated Herpesvirus Cloned in a Bacterial Artificial Chromosome: Application for Genetic Analysis

Journal of Virology ◽

10.1128/jvi.76.12.6185-6196.2002 ◽

2002 ◽

Vol 76 (12) ◽

pp. 6185-6196 ◽

Cited By ~ 185

Author(s):

Fu-Chun Zhou ◽

Yan-Jin Zhang ◽

Jian-Hong Deng ◽

Xin-Ping Wang ◽

Hong-Yi Pan ◽

...

Keyword(s):

Genetic Analysis ◽

Cell Lines ◽

Kaposi's Sarcoma ◽

Kaposi’S Sarcoma ◽

Artificial Chromosome ◽

Lytic Replication ◽

Kshv Infection ◽

293 Cells ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma and several other malignancies. The lack of an efficient infection system has impeded the understanding of KSHV-related pathogenesis. A genetic approach was used to isolate infectious KSHV. Recombinant bacteria artificial chromosome (BAC) KSHV containing hygromycin resistance and green fluorescent protein (GFP) markers was generated by homologous recombination in KSHV-infected BCBL-1 cells. Recombinant KSHV genomes from cell clones that were resistant to hygromycin, expressed GFP, and produced infectious virions after induction with tetradecanoyl phorbol acetate (TPA) were rescued in Escherichia coli and reconstituted in 293 cells. Several 293 cell lines resulting from infection with recombinant virions induced from a full-length recombinant KSHV genome, named BAC36, were obtained. BAC36 virions established stable latent infection in 293 cells, harboring 1 to 2 copies of viral genome per cell and expressing viral latent proteins, with ≈0.5% of cells undergoing spontaneous lytic replication, which is reminiscent of KSHV infection in Kaposi's sarcoma tumors. TPA treatment induced BAC36-infected 293 cell lines into productive lytic replication, expressing lytic proteins and producing virions that efficiently infected normal 293 cells with a ≈50% primary infection rate. BAC36 virions were also infectious to HeLa and E6E7-immortalized human endothelial cells. Since BAC36 can be efficiently shuttled between bacteria and mammalian cells, it is useful for KSHV genetic analysis. The feasibility of the system was illustrated through the generation of a KSHV mutant with the vIRF gene deleted. This cellular model is useful for the investigation of KSHV infection and pathogenesis.

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In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi's sarcoma-associated herpesvirus-like (KSHV) DNA sequences

Blood ◽

10.1182/blood.v86.7.2708.bloodjournal8672708 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2708-2714 ◽

Cited By ~ 23

Author(s):

E Cesarman ◽

PS Moore ◽

PH Rao ◽

G Inghirami ◽

DM Knowles ◽

...

Keyword(s):

Cell Lines ◽

Acquired Immunodeficiency Syndrome ◽

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Acquired Immunodeficiency ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Hodgkin's Lymphomas ◽

Immunodeficiency Syndrome ◽

Kaposi’S Sarcoma Associated Herpesvirus

Two unique DNA fragments were recently identified in over 90% of acquired immunodeficiency syndrome (AIDS)-related Kaposi's sarcoma tissues. Sequence analysis suggests that these fragments belong to a previously unidentified human herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). These fragments have also been identified in a subset of non-Hodgkin's lymphomas in human immunodeficiency virus-positive patients; specifically, in body cavity-based lymphomas (AIDS-BCBLs). We have established two cell lines derived from AIDS-BCBLs, BC-1 and BC-2, which retain the KSHV sequences, and have used them to further characterize this putative viral genome. In this report, we demonstrate that the KSHV sequences represent a portion of a much larger DNA molecule that is located predominantly in the nucleus of the infected cells. In situ hybridization of metaphase spreads made from both of these cell lines show these sequences in episomal structures. Their presence in the cells as large nuclear episomes supports previous sequence homology data suggesting that KSHV belongs to the herpesvirus family. These cell lines provide a continuous source of KSHV sequences. Thus, they represent an important tool for future studies of this recently described human herpesvirus that may be involved in the pathogenesis of Kaposi's sarcoma and some AIDS-related non-Hodgkin's lymphomas.

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K15 Protein of Kaposi’s Sarcoma-Associated Herpesvirus Is Latently Expressed and Binds to HAX-1, a Protein with Antiapoptotic Function

Journal of Virology ◽

10.1128/jvi.76.2.802-816.2002 ◽

2002 ◽

Vol 76 (2) ◽

pp. 802-816 ◽

Cited By ~ 148

Author(s):

Tyson V. Sharp ◽

Hsei-Wei Wang ◽

Andrew Koumi ◽

Daniel Hollyman ◽

Yoshio Endo ◽

...

Keyword(s):

Cell Lines ◽

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Regulatory Protein ◽

Ectopic Expression ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The Kaposi’s sarcoma-associated herpesvirus (KSHV) (or human herpesvirus 8) open reading frame (ORF) K15 encodes a putative integral transmembrane protein in the same genomic location as latent membrane protein 2A of Epstein-Barr virus. Ectopic expression of K15 in cell lines revealed the presence of several different forms ranging in size from full length, ∼50 kDa, to 17 kDa. Of these different species the 35- and 23-kDa forms were predominant. Mutational analysis of the initiator AUG indicated that translation initiation from this first AUG is required for K15 expression. Computational analysis indicates that the different forms detected may arise due to proteolytic cleavage at internal signal peptide sites. We show that K15 is latently expressed in KSHV-positive primary effusion lymphoma cell lines and in multicentric Castleman’s disease. Using a yeast two-hybrid screen we identified HAX-1 (HS1 associated protein X-1) as a binding partner to the C terminus of K15 and show that K15 interacts with cellular HAX-1 in vitro and in vivo. Furthermore, HAX-1 colocalizes with K15 in the endoplasmic reticulum and mitochondria. The function of HAX-1 is unknown, although the similarity of its sequence to those of Nip3 and Bcl-2 infers a role in the regulation of apoptosis. We show here that HAX-1 can form homodimers in vivo and is a potent inhibitor of apoptosis and therefore represents a new apoptosis regulatory protein. The putative functions of K15 with respect to its interaction with HAX-1 are discussed.

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In vitro establishment and characterization of two acquired immunodeficiency syndrome-related lymphoma cell lines (BC-1 and BC-2) containing Kaposi's sarcoma-associated herpesvirus-like (KSHV) DNA sequences

Blood ◽

10.1182/blood.v86.7.2708.2708 ◽

1995 ◽

Vol 86 (7) ◽

pp. 2708-2714 ◽

Cited By ~ 407

Author(s):

E Cesarman ◽

PS Moore ◽

PH Rao ◽

G Inghirami ◽

DM Knowles ◽

...

Keyword(s):

Cell Lines ◽

Acquired Immunodeficiency Syndrome ◽

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Acquired Immunodeficiency ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Hodgkin's Lymphomas ◽

Immunodeficiency Syndrome ◽

Kaposi’S Sarcoma Associated Herpesvirus

Abstract Two unique DNA fragments were recently identified in over 90% of acquired immunodeficiency syndrome (AIDS)-related Kaposi's sarcoma tissues. Sequence analysis suggests that these fragments belong to a previously unidentified human herpesvirus, Kaposi's sarcoma-associated herpesvirus (KSHV). These fragments have also been identified in a subset of non-Hodgkin's lymphomas in human immunodeficiency virus-positive patients; specifically, in body cavity-based lymphomas (AIDS-BCBLs). We have established two cell lines derived from AIDS-BCBLs, BC-1 and BC-2, which retain the KSHV sequences, and have used them to further characterize this putative viral genome. In this report, we demonstrate that the KSHV sequences represent a portion of a much larger DNA molecule that is located predominantly in the nucleus of the infected cells. In situ hybridization of metaphase spreads made from both of these cell lines show these sequences in episomal structures. Their presence in the cells as large nuclear episomes supports previous sequence homology data suggesting that KSHV belongs to the herpesvirus family. These cell lines provide a continuous source of KSHV sequences. Thus, they represent an important tool for future studies of this recently described human herpesvirus that may be involved in the pathogenesis of Kaposi's sarcoma and some AIDS-related non-Hodgkin's lymphomas.

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Lytic Replication-Defective Kaposi's Sarcoma-Associated Herpesvirus: Potential Role in Infection and Malignant Transformation

Journal of Virology ◽

10.1128/jvi.78.20.11108-11120.2004 ◽

2004 ◽

Vol 78 (20) ◽

pp. 11108-11120 ◽

Cited By ~ 14

Author(s):

Jian-Hong Deng ◽

Yan-Jin Zhang ◽

Xin-Ping Wang ◽

Shou-Jiang Gao

Keyword(s):

Malignant Transformation ◽

Cell Lines ◽

Kaposi's Sarcoma ◽

Potential Role ◽

Primary Effusion Lymphoma ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Screening Methods ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G0/G1 apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.

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Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing

Journal of Virology ◽

10.1128/jvi.00411-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8225-8235 ◽

Cited By ~ 70

Author(s):

Hyun Jin Kwun ◽

Suzane Ramos da Silva ◽

Ishita M. Shah ◽

Neil Blake ◽

Patrick S. Moore ◽

...

Keyword(s):

Antigen Processing ◽

Kaposi's Sarcoma ◽

Epstein Barr Virus ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

Barr Virus ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Epstein Barr ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses.

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Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus

Journal of Virology ◽

10.1128/jvi.75.3.1378-1386.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1378-1386 ◽

Cited By ~ 134

Author(s):

Jeffrey Vieira ◽

Patricia O'Hearn ◽

Louise Kimball ◽

Bala Chandran ◽

Lawrence Corey

Keyword(s):

Human Cytomegalovirus ◽

Kaposi's Sarcoma ◽

Human Fibroblasts ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Lytic Cycle ◽

Nuclear Antigen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) andneo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.

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Mechanism of Angiopoietin-1 Upregulation in Kaposi's Sarcoma-Associated Herpesvirus-Infected PEL Cell Lines

Journal of Virology ◽

10.1128/jvi.03144-14 ◽

2015 ◽

Vol 89 (9) ◽

pp. 4786-4797 ◽

Cited By ~ 2

Author(s):

Xin Zheng ◽

Eriko Ohsaki ◽

Keiji Ueda

Keyword(s):

Cell Lines ◽

Kaposi's Sarcoma ◽

Primary Effusion Lymphoma ◽

Regulatory Region ◽

Leukemia Cell ◽

Kaposi’S Sarcoma ◽

Dependent Manner ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus ◽

Angiopoietin 1

ABSTRACTAngiopoietin-1 (ANGPT-1) is a secreted glycoprotein that was first characterized as a ligand of the Tie2 receptor. In a previous study using microarray analysis, we found that the expression of ANGPT-1 was upregulated in Kaposi's sarcoma-associated herpesvirus (KSHV)-infected primary effusion lymphoma (PEL) cell lines compared with that in uninfected Burkitt and other leukemia cell lines. Other authors have also reported focal expression of ANGPT-1 mRNA in biopsy specimens of Kaposi's sarcoma (KS) tissue from patients with AIDS. Here, to confirm these findings, we examined the expression and secretion levels of ANGPT-1 in KSHV-infected PEL cell lines and address the mechanisms ofANGPT-1transcriptional regulation. We also showed that ANGPT-1 was expressed and localized in the cytoplasm and secreted into the supernatant of KSHV-infected PEL cells. Deletion studies of the regulatory region revealed that the region encompassing nucleotides −143 to −125 of theANGPT-1-regulating sequence was responsible for this upregulation. Moreover, an electrophoretic mobility shift assay and chromatin immunoprecipitation, followed by quantitative PCR, suggested that some KSHV-infected PEL cell line-specific DNA-binding factors, such as OCT-1, should be involved in the upregulation ofANGPT-1in a sequence-dependent manner.IMPORTANCEWe confirmed that ANGPT-1 was expressed in and secreted from KSHV-infected PEL cells and that the transcriptional activity ofANGPT-1was upregulated. A 19-bp fragment was identified as the region responsible forANGPT-1upregulation through binding with OCT-1 as a core factor in PEL cells. This study suggests that ANGPT-1 is overproduced in KSHV-infected PEL cells, which could affect the pathophysiology of AIDS patients with PEL.

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