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Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

ABSTRACT: We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of herpesvirus saimiri. KSHV sVCA was expressed from a 0.85-kb mRNA present late in lytic KSHV replication in BC-1 cells. This transcript was sensitive to phosphonoacetic acid and phosphonoformic acid, inhibitors of herpesvirus DNA replication. KSHV sVCA expressed in mammalian cells or Escherichia coli or translated in vitro was recognized as an antigen by antisera from KS patients. Rabbit antisera raised to KSHV sVCA expressed in E. coli detected a 22-kDa protein in KSHV-infected human B cells. Overexpressed KSHV sVCA purified from E. coli and used as an antigen in immunoblot screening assay did not cross-react with EBV BFRF3. Antibodies to sVCA were present in 89% of 47 human immunodeficiency virus (HIV)-positive patients with KS, in 20% of 54 HIV-positive patients without KS, but in none of 122 other patients including children born to HIV-seropositive mothers and patients with hemophilia, autoimmune disease, or nasopharyngeal carcinoma. Low-titer antibody was detected in three sera from 28 healthy subjects. Antibodies to recombinant sVCA correlate with KS in high-risk populations. Recombinant sVCA can be used to examine the seroepidemiology of infection with KSHV in the general population.

SUBMITTER: Lin SF

PROVIDER: S-EPMC191437 | biostudies-other | 1997 Apr

REPOSITORIES: biostudies-other

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Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

Lin S F SF Sun R R Heston L L Gradoville L L Shedd D D Haglund K K Rigsby M M Miller G G

Journal of virology 19970401 4

We describe a recombinant antigen for use in serologic tests for antibodies to Kaposi's sarcoma (KS)-associated herpesvirus (KSHV). The cDNA for a small viral capsid antigen (sVCA) was identified by immunoscreening of a library prepared from the BC-1 body cavity lymphoma cell line induced into KSHV lytic gene expression by sodium butyrate. The cDNA specified a 170-amino-acid peptide with homology to small viral capsid proteins encoded by the BFRF3 gene of Epstein-Barr virus and the ORF65 gene of ...[more]

PMID: 9060668

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Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentric Castleman disease (MCD). The KSHV life cycle has two principal gene repertoires, latent and lytic. KSHV viral interleukin-6 (vIL-6), an analog of human IL-6, is usually lytic; production of vIL-6 by involved plasmablasts is a central feature of KSHV-MCD. vIL-6 also plays a role in PEL and KS. We show that a number of plasmablasts from lymph nodes of patients with KSHV-MCD express vIL-6 but not ORF45, a KSHV lytic gene. We further show that vIL-6 is directly induced by the spliced (active) X-box binding protein-1 (XBP-1s), a transcription factor activated by endoplasmic reticulum (ER) stress and differentiation of B cells in lymph nodes. The promoter region of vIL-6 contains several potential XBP-response elements (XREs), and two of these elements in particular mediate the effect of XBP-1s. Mutation of these elements abrogates the response to XBP-1s but not to the KSHV replication and transcription activator (RTA). Also, XBP-1s binds to the vIL-6 promoter in the region of these XREs. Exposure of PEL cells to a chemical inducer of XBP-1s can induce vIL-6. Patient-derived PEL tumor cells that produce vIL-6 frequently coexpress XBP-1, and immunofluorescence staining of involved KSHV-MCD lymph nodes reveals that most plasmablasts expressing vIL-6 also coexpress XBP-1. These results provide evidence that XBP-1s is a direct activator of KSHV vIL-6 and that this is an important step in the pathogenesis of KSHV-MCD and PEL.Kaposi sarcoma herpesvirus (KSHV)-associated multicentric Castleman disease (KSHV-MCD) is characterized by severe inflammatory symptoms caused by an excess of cytokines, particularly KSHV-encoded viral interleukin-6 (vIL-6) produced by lymph node plasmablasts. vIL-6 is usually a lytic gene. We show that a number of KSHV-MCD lymph node plasmablasts express vIL-6 but do not have full lytic KSHV replication. Differentiating lymph node B cells express spliced (active) X-box binding protein-1 (XBP-1s). We show that XBP-1s binds to the promoter of vIL-6 and can directly induce production of vIL-6 through X-box protein response elements on the vIL-6 promoter region. We further show that chemical inducers of XBP-1s can upregulate production of vIL-6. Finally, we show that most vIL-6-producing plasmablasts from lymph nodes of KSHV-MCD patients coexpress XBP-1s. These results demonstrate that XBP-1s can directly induce vIL-6 and provide evidence that this is a key step in the pathogenesis of KSHV-MCD and other KSHV-induced diseases.

Project description:UnlabelledCapsid-associated tegument proteins have been identified in alpha- and betaherpesviruses to play an essential role in viral DNA packaging. Whether and how such tegument proteins exist in gammaherpesviruses have been mysteries. Here, we report a 6-Å-resolution cryo-electron microscopy (cryo-EM) structure of Kaposi's sarcoma-associated herpesvirus (KSHV) virion, a member of the oncogenic gammaherpesvirus subfamily. The KSHV virion structure reveals, for the first time, how capsid-associated tegument proteins are organized in a gammaherpesvirus, with five tegument densities capping each penton vertex, a pattern highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Each KSHV tegument density can be divided into three prominent regions: a penton-binding globular region, a helix-bundle stalk region, and a β-sheet-rich triplex-binding region. Fitting of the crystal structure of the truncated HSV-1 UL25 protein (the KSHV ORF19 homolog) and secondary structure analysis of the full-length ORF19 established that ORF19 constitutes the globular region with an N-terminal, 60-amino-acid-long helix extending into the stalk region. Matching secondary structural features resolved in the cryo-EM density with secondary structures predicted by sequence analysis identifies the triplex-binding region to be ORF32, a homolog of alphaherpesvirus UL17. Despite the high level of tegument structural similarities between KSHV and alphaherpesvirus, an ORF19 monomer in KSHV, in contrast to a UL25 dimer in alphaherpesviruses, binds each penton subunit, an observation that correlates with conformational differences in their pentons. This newly discovered organization of triplex-ORF32-ORF19 also resolves a long-standing mystery surrounding the virion location and conformation of alphaherpesvirus UL25 protein.ImportanceSeveral capsid-associated tegument proteins have been identified in the alpha- and betaherpesvirus subfamilies of the Herpesviridae. These tegument proteins play essential roles in viral propagation and are potential drug targets for curbing herpesvirus infections. However, no such tegument proteins have been identified for gammaherpesviruses, the third herpesvirus subfamily, which contains members causing several human cancers. Here, by high-resolution cryo-EM, we show the three-dimensional structure of the capsid-associated tegument proteins in the prototypical member of gammaherpesviruses, KSHV. The cryo-EM structure reveals that the organization of KSHV capsid-associated tegument proteins is highly similar to that in alphaherpesvirus but completely different from that in betaherpesvirus. Structural analyses further localize ORF19 and ORF32 proteins (the alphaherpesvirus UL25 and UL17 homologs in KSHV, respectively) in the KSHV capsid-associated tegument cryo-EM structure. These findings also resolve a long-standing mystery regarding the location and conformation of alphaherpesvirus UL25 protein inside the virion.

Dataset Information

Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

Publications

Identification, expression, and immunogenicity of Kaposi's sarcoma-associated herpesvirus-encoded small viral capsid antigen.

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