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Multiple defects, including premature apoptosis, prevent Kaposi's sarcoma-associated herpesvirus replication in murine cells.

ABSTRACT: The development of a mouse model for Kaposi's sarcoma-associated herpesvirus (KSHV) infection has been impeded by the limited host range of the virus. Here, we have examined the molecular basis of this host range restriction. KSHV efficiently enters murine cells and establishes latency. However, ectopic expression of the lytic switch protein RTA (replication and transcription activator) in these cells induces little viral gene expression and no virus production. Upon treatment with histone deacetylase inhibitors, KSHV-infected murine cells display more extensive but aberrant viral transcription and do not support either viral DNA synthesis or the production of infectious virions. These aberrantly infected cells also display markedly enhanced apoptosis. Genetic ablation of the mitochondrial apoptotic pathway in these cells prolongs their survival and permits viral DNA replication but does not rescue the generation of virions. We conclude that multiple defects, both prior to and following DNA synthesis, restrict lytic KSHV infection in murine cells.

SUBMITTER: Austgen K

PROVIDER: S-EPMC3264352 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Multiple defects, including premature apoptosis, prevent Kaposi's sarcoma-associated herpesvirus replication in murine cells.

Austgen Kathryn K Oakes Scott A SA Ganem Don D

Journal of virology 20111130 3

The development of a mouse model for Kaposi's sarcoma-associated herpesvirus (KSHV) infection has been impeded by the limited host range of the virus. Here, we have examined the molecular basis of this host range restriction. KSHV efficiently enters murine cells and establishes latency. However, ectopic expression of the lytic switch protein RTA (replication and transcription activator) in these cells induces little viral gene expression and no virus production. Upon treatment with histone deace ...[more]

PMID: 22130538

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Project description:Molecular mechanisms of Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation have been studied primarily by measuring the total or average activity of an infected cell population, which often consists of a mixture of both nonresponding and reactivating cells that in turn contain KSHVs at various stages of replication. Studies on KSHV gene regulation at the individual cell level would allow us to better understand the basis for this heterogeneity, and new preventive measures could be developed based on findings from nonresponding cells exposed to reactivation stimuli. Here, we generated a recombinant reporter virus, which we named "Rainbow-KSHV," that encodes three fluorescence-tagged KSHV proteins (mBFP2-ORF6, mCardinal-ORF52, and mCherry-LANA). Rainbow-KSHV replicated similarly to a prototype reporter-KSHV, KSHVr.219, and wild-type BAC16 virus. Live imaging revealed unsynchronized initiation of reactivation and KSHV replication with diverse kinetics between individual cells. Cell fractionation revealed temporal gene regulation, in which early lytic gene expression was terminated in late protein-expressing cells. Finally, isolation of fluorescence-positive cells from nonresponders increased dynamic ranges of downstream experiments 10-fold. Thus, this study demonstrates a tool to examine heterogenic responses of KSHV reactivation for a deeper understanding of KSHV replication.IMPORTANCE Sensitivity and resolution of molecular analysis are often compromised by the use of techniques that measure the ensemble average of large cell populations. Having a research tool to nondestructively identify the KSHV replication stage in an infected cell would not only allow us to effectively isolate cells of interest from cell populations but also enable more precise sample selection for advanced single-cell analysis. We prepared a recombinant KSHV that can report on its replication stage in host cells by differential fluorescence emission. Consistent with previous host gene expression studies, our experiments reveal the highly heterogenic nature of KSHV replication/gene expression at individual cell levels. The utilization of a newly developed reporter-KSHV and initial characterization of KSHV replication in single cells are presented.

Project description:The anaphase-promoting complex, or cyclosome (APC/C), is a large E3 ubiquitin ligase composed of 14 subunits. The activity of APC/C oscillates during the cell cycle to ensure a timely transition through each phase by promoting the degradation of important cell cycle regulators. Of the human herpesviruses, cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) both impair the activity of APC/C during their lytic replication cycle through virus-encoded protein kinases. Here, we addressed whether the oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV) deregulates the activity of APC/C during the lytic replication cycle. To this end, we used the well-characterized iSLK.219 cell model of KSHV infection and established a new infection model of primary lymphatic endothelial cells (LECs) infected with a lytically replicating KSHV BAC16 mutant. In contrast to those of EBV and HCMV, the KSHV lytic cycle occurs while the APC/C is active. Moreover, interfering with the activity of APC/C did not lead to major changes in the production of infectious virus. We further investigated whether rereplication stress induced by the unscheduled activation of the APC/C-CDH1 complex affects the number and integrity of KSHV viral episomes. Deep sequencing of the viral episomes and host chromosomes in iSLK.219 cells revealed that, while distinct regions in the cellular chromosomes were severely affected by rereplication stress, the integrity of the viral episomes remained unaltered.IMPORTANCE DNA viruses have evolved complex strategies to gain control over the cell cycle. Several of them target APC/C, a key cellular machinery that controls the timely progression of the cell cycle, by either blocking or enhancing its activity. Here, we investigated the activity of APC/C during the lytic replication cycle of KSHV and found that, in contrast to that of KSHV's close relatives EBV and HCMV, KSHV lytic replication occurs while the APC/C is active. Perturbing APC/C activity by depleting a core protein or the adaptor proteins of the catalytic domain, and hence interfering with normal cell-cycle progression, did not affect virus replication. This suggests that KSHV has evolved to replicate independently of the activity of APC/C and in various cell cycle conditions.

Dataset Information

Multiple defects, including premature apoptosis, prevent Kaposi's sarcoma-associated herpesvirus replication in murine cells.

Publications

Multiple defects, including premature apoptosis, prevent Kaposi's sarcoma-associated herpesvirus replication in murine cells.

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