Inhibition of the lytic cycle of Kaposi's sarcoma-associated herpesvirus by cohesin factors following de novo infection.
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ABSTRACT:
SUBMITTER: Toth Z
PROVIDER: S-EPMC5653454 | biostudies-literature | 2017 Dec
REPOSITORIES: biostudies-literature
Publications
Inhibition of the lytic cycle of Kaposi's sarcoma-associated herpesvirus by cohesin factors following de novo infection.
Toth Zsolt Z Smindak Richard J RJ Papp Bernadett B
Virology 20171201
Establishment of Kaposi's sarcoma-associated herpesvirus (KSHV) latency following infection is a multistep process, during which polycomb proteins are recruited onto the KSHV genome, which is crucial for the genome-wide repression of lytic genes during latency. Strikingly, only a subset of lytic genes are expressed transiently in the early phase of infection prior to the binding of polycomb proteins onto the KSHV genome, which raises the question what restricts lytic gene expression in the first ...[more]
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Project description:CTCF and the cohesin complex modify chromatin by binding to DNA and interacting with each other and with other cellular proteins. Both proteins regulate transcription by a variety of local effects on transcription and by long-range topological effects. CTCF and cohesin also bind to herpesvirus genomes at specific sites and regulate viral transcription during latent and lytic cycles of replication. Kaposi's sarcoma-associated herpesvirus (KSHV) transcription is regulated by CTCF and cohesin, with both proteins previously reported to act as restrictive factors for lytic cycle transcription and virion production. In this study, we examined the interdependence of CTCF and cohesin binding to the KSHV genome. Chromatin immunoprecipitation sequencing (ChIP-seq) analyses revealed that cohesin binding to the KSHV genome is highly CTCF dependent, whereas CTCF binding does not require cohesin. Furthermore, depletion of CTCF leads to the almost complete dissociation of cohesin from sites at which they colocalize. Thus, previous studies that examined the effects of CTCF depletion actually represent the concomitant depletion of both CTCF and cohesin components. Analysis of the effects of single and combined depletion indicates that CTCF primarily activates KSHV lytic transcription, whereas cohesin has primarily inhibitory effects. Furthermore, CTCF or cohesin depletion was found to have regulatory effects on cellular gene expression relevant for the control of viral infection, with both proteins potentially facilitating the expression of multiple genes important in the innate immune response to viruses. Thus, CTCF and cohesin have both positive and negative effects on KSHV lytic replication as well as effects on the host cell that enhance antiviral defenses.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is causally linked to Kaposi's sarcoma and several lymphoproliferative diseases. KSHV, like other herpesviruses, intermittently reactivates from latency and enters a lytic cycle in which numerous lytic mRNAs and proteins are produced, culminating in infectious virion production. These lytic proteins may also contribute to tumorigenesis. Reactivation from latency is controlled by processes that restrict or activate the transcription of KSHV lytic genes. KSHV gene expression is modulated by binding of the host cell proteins CTCF and cohesin complex to the KSHV genome. These proteins bind to and modulate the conformation of chromatin, thereby regulating transcription. We have analyzed the interdependence of binding of CTCF and cohesin and demonstrate that while CTCF is required for cohesin binding to KSHV, they have very distinct effects, with cohesin primarily restricting KSHV lytic transcription. Furthermore, we show that cohesin and CTCF also exert effects on the host cell that promote antiviral defenses.
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Project description:The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system is an RNA-guided, DNA editing method that has been widely used for gene editing, including human viruses. Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), following latent infection in human cells, can cause a variety of malignancies, such as Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD), with a high prevalence in immunocompromised patients. Of significant concern, the latent infection with KSHV has been shown to lead to increased resistance to antiviral therapies. MicroRNAs (miRNAs) are a set of non-coding, small RNA molecules that regulate protein-coding genes at the post-transcriptional and translational levels. KSHV has its miRNAs, most of which are expressed in latently infected cells and play a crucial role in maintaining KSHV latency. Notably, by regulating the expression of the downstream target genes in host cells, KSHV miRNAs can interact with the host environment to promote the development of KSHV-related diseases. Although CRISPR/Cas9 has been reported to edit KSHV protein-coding genes, there is no published literature on whether the CRISPR/Cas9 system can regulate the expression of KSHV miRNAs. In this study, we used CRISPR/Cas9 to inhibit the expression of KSHV miRNAs by directly editing the DNA sequences of individual KSHV miRNAs, or the promoter of clustered KHSV miRNAs, in latent KSHV-infected PEL cells. Our results show that CRISPR/Cas9 can ablate KSHV miRNAs expression, which in turn leads to the upregulation of viral lytic genes and alteration of host cellular gene expression. To the best of our knowledge, our study is the first reported demonstration of the CRISPR/Cas9 system editing KSHV miRNAs, further expanding the application of CRISPR/Cas9 as a novel antiviral strategy targeting KSHV latency.
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