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Polymerase chain reaction detection of Kaposi's sarcoma-associated herpesvirus-optimized protocols and their application to myeloma.

ABSTRACT: Since its discovery in 1994, KSHV (also called human herpesvirus-8 or HHV8) has been implicated in a variety of disorders. Although the association of KSHV with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease has been well established, its presence in some other diseases, such as multiple myeloma, remains controversial. Because most KSHV studies are based on polymerase chain reaction (PCR) analysis, the conflicting data may be attributable to variations in the methods, primer sets, and target sequences selected. To establish an efficient and reliable PCR approach for KSHV detection we designed eight sets of primers to six regions (ORFK1, ORFK2, ORFK9, ORK26, ORF72, and ORF74) of the KSHV genome using appropriate database and software. The detection sensitivity of these primers was carefully assessed and their reliability was strictly validated in a series of positive (15 KS and PEL samples) and negative (16 lymphoid tissues) controls. We found that primer sets to the ORFK9 region showed the highest sensitivity, whereas primer sets to ORFK1 and ORF74 showed the lowest sensitivity. Primer sets to ORFK9, ORF26 and ORF72 regions detected all of the positive cases, whereas other primer sets showed varying detection rates or nonspecific bands. All 16 negative controls were negative with all primer sets. However, six of 16 negative controls became positive when we used nested PCR targeting ORF26. Therefore, multiple target KSHV sequences increase the detection efficiency, while nested PCR protocols are likely to introduce false positivity. Using ORFK9, ORF26 and ORF72 primer sets, we screened bone marrow biopsies from 18 cases of multiple myeloma, and failed to detect any KSHV sequences. This finding supports the conclusion that KSHV is not associated with multiple myeloma. Indeed, our results further confirm that although KSHV is universally present in Kaposi's sarcoma and primary effusion lymphoma, it is not ubiquitious.

SUBMITTER: Pan L

PROVIDER: S-EPMC1907348 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Polymerase chain reaction detection of Kaposi's sarcoma-associated herpesvirus-optimized protocols and their application to myeloma.

Pan L L Milligan L L Michaeli J J Cesarman E E Knowles D M DM

The Journal of molecular diagnostics : JMD 20010201 1

Since its discovery in 1994, KSHV (also called human herpesvirus-8 or HHV8) has been implicated in a variety of disorders. Although the association of KSHV with Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease has been well established, its presence in some other diseases, such as multiple myeloma, remains controversial. Because most KSHV studies are based on polymerase chain reaction (PCR) analysis, the conflicting data may be attributable to variatio ...[more]

PMID: 11227070

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Project description:Locally concentrated nuclear factors ensure efficient binding to DNA templates, facilitating RNA polymerase II recruitment and frequent reutilization of stable preinitiation complexes. We have uncovered a mechanism for effective viral transcription by focal assembly of RNA polymerase II around Kaposi's sarcoma-associated herpesvirus (KSHV) genomes in the host cell nucleus. Using immunofluorescence labeling of latent nuclear antigen (LANA) protein, together with fluorescence in situ RNA hybridization (RNA-FISH) of the intron region of immediate early transcripts, we visualized active transcription of viral genomes in naturally infected cells. At the single-cell level, we found that not all episomes were uniformly transcribed following reactivation stimuli. However, those episomes that were being transcribed would spontaneously aggregate to form transcriptional "factories," which recruited a significant fraction of cellular RNA polymerase II. Focal assembly of "viral transcriptional factories" decreased the pool of cellular RNA polymerase II available for cellular gene transcription, which consequently impaired cellular gene expression globally, with the exception of selected ones. The viral transcriptional factories localized with replicating viral genomic DNAs. The observed colocalization of viral transcriptional factories with replicating viral genomic DNA suggests that KSHV assembles an "all-in-one" factory for both gene transcription and DNA replication. We propose that the assembly of RNA polymerase II around viral episomes in the nucleus may be a previously unexplored aspect of KSHV gene regulation by confiscation of a limited supply of RNA polymerase II in infected cells.IMPORTANCE B cells infected with Kaposi's sarcoma-associated herpesvirus (KSHV) harbor multiple copies of the KSHV genome in the form of episomes. Three-dimensional imaging of viral gene expression in the nucleus allows us to study interactions and changes in the physical distribution of these episomes following stimulation. The results showed heterogeneity in the responses of individual KSHV episomes to stimuli within a single reactivating cell; those episomes that did respond to stimulation, aggregated within large domains that appear to function as viral transcription factories. A significant portion of cellular RNA polymerase II was trapped in these factories and served to transcribe viral genomes, which coincided with an overall decrease in cellular gene expression. Our findings uncover a strategy of KSHV gene regulation through focal assembly of KSHV episomes and a molecular mechanism of late gene expression.

Project description:Retinoic acid-inducible gene I (RIG-I) is a cytosolic pathogen recognition receptor that initiates the innate immune response against many RNA viruses. We previously showed that RIG-I restricts Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation (J. A. West et al., J Virol 88:5778-5787, 2014, https://doi.org/10.1128/JVI.03226-13). In this study, we report that KSHV stimulates the RIG-I signaling pathway in a RNA polymerase (Pol) III-independent manner and subsequently induces type I interferon (IFN) responses. Knockdown or inhibition of RNA Pol III had no effect on beta interferon (IFN-?) induction by KSHV. By using high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) approach, we identified multiple KSHV regions that give rise to RNA fragments binding to RIG-I, such as ORF810420-10496, Repeat region (LIR1)119059-119204, and ORF2543561-43650 The sequence dissimilarity between these fragments suggests that RIG-I detects a particular structure rather than a specific sequence motif. Synthesized ORF810420-10496 RNA stimulated RIG-I-dependent but RNA Pol III-independent IFN-? signaling. In summary, several KSHV RNAs are sensed by RIG-I in a RNA Pol III-independent manner.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Innate immune responses against viral infections, especially the induction of type I interferon, are critical for limiting the replication of viruses. Retinoic acid-inducible gene I (RIG-I), a cytosolic RNA helicase sensor, plays a significant role in the induction of type I interferon responses following viral infection. Here, we identified multiple RNA regions in KSHV as potential virus ligands that bind to RIG-I and stimulate RIG-I-dependent but RNA Pol III-independent IFN-? signaling. Our results expand the role of RIG-I by providing an example of a DNA virus activating a canonical RNA-sensing pathway.

Dataset Information

Polymerase chain reaction detection of Kaposi's sarcoma-associated herpesvirus-optimized protocols and their application to myeloma.

Publications

Polymerase chain reaction detection of Kaposi's sarcoma-associated herpesvirus-optimized protocols and their application to myeloma.

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