Mesri, E. A. | Search bioRxiv

bioRxiv2019

A role of hypoxia inducible factor 1 alpha in Mouse Gammaherpesvirus 68 (MHV68) lytic replication and reactivation from latency

The hypoxia inducible factor 1 alpha (HIFI) protein and the hypoxic microenvironment are critical for infection and pathogenesis by the oncogenic gammaherpesviruses ({gamma}HV) such as Kaposi Sarcoma-associated Herpes Virus (KSHV) and Epstein-Barr virus (EBV). However, understanding the role of HIFI during the virus life cycle and its biological relevance in the context of host pathogenesis has been challenging due to the lack of animal models for human {gamma}HV. To study the role of HIFI we employed the murine gammaherpesvirus 68 (MHV68), a rodent pathogen that readily infects laboratory mice. We show that MHV68 infection induces HIFI protein and HIFI-responsive gene expression in permissive cells. Deletion of HIFI reduces virus production due to a global downregulation of viral gene expression. Most notable was the marked decrease in many viral genes bearing hypoxia regulatory element (HRE) such as viral G-Protein Coupled Receptor (vGPCR), which is known to activate HIF1 transcriptional activity during KSHV infection. Intranasal infection of HIF1LoxP/LoxP mice with MHV68 expressing Cre-recombinase impaired virus expansion during early acute infection and affected lytic reactivation in the splenocytes explanted from mice. Moreover, low oxygen conditions accelerated lytic reactivation and enhanced virus production in MHV68 infected splenocytes. Thus, we conclude that HIFI plays a critical role to promote virus replication. Our results highlight the importance of the mutual interactions of the oxygen-sensing machinery and gammaherpesviruses in viral replication and pathogenesis.\n\nAUTHOR SUMMARYThe host oxygen sensing machinery including the HIF1 pathway is important during the viral life cycle of oncogenic gammaherpesviruses such as KSHV and EBV. However, due to the host specificity, the effects of HIF1 in herpes biology is limited to studies with in vitro systems. Here, we study the role of HIF1 using the mouse gammaherpesvirus 68 (MHV68) that readily infects laboratory mice. We demonstrate that MHV68 infection upregulates HIF1 during replication and inactivation of HIF1 transcriptional activity significantly decreased viral genes expression which results in impaired virus production in vitro. In vivo deletion of HIF1 impaired viral expansion during acute infection and affected reactivation from latency. These results show the importance of the interplay with the oxygen-sensing machinery in gammaherpesvirus infection and pathogenesis, placing the MHV68 mouse model as a unique platform to gain insight into this important aspect of oncogenic gamma-herpesviruses biology and to test HIF1 targeted therapeutics.

cancer biology↗

bioRxiv2019

RNA-sequencing analysis of a multistep and hit-and-run cell and animal model of KSHV tumorigenesis reveal the roles of mutations, CpG methylation, and viral-infection footprints in oncogenesis.

Human viral oncogenesis is the consequence of cell transformation mediated by virally encoded oncogenes in combination with host oncogenic alterations. Kaposis sarcoma (KS), caused by the Kaposis sarcoma-associated herpes virus (KSHV), is an AIDS-associated cancer characterized by angiogenesis and spindle-cells proliferation. KSHV-infected KS lesions are composed of latently-infected cells, as well as cells expressing lytic genes that have been implicated in the development of the KS angioproliferative phenotype. The existence of KS lesions with varying levels of KSHV-infected cells suggests also the existence of virus-independent \"hit-and-run\" mechanisms of sarcomagenesis, whereby viral infection irreversibly induce genetic or epigenetic oncogenic alterations in host cells. We have integrated genetic mutations, changes in expression signatures and methylation analysis to dissect genetic and epigenetic signaling pathways in an unbiased manner in the mECK36 mouse model of KSHV tumorigenesis. Pathway analysis of differential expressed genes (DEGs) showed KSHV lytic switch, DNA methylation and Epigenetic as the most regulated pathways during KSHV-dependent in vivo tumorigenesis. Methylation analysis data indicates that during the development of KSHV-infected tumors the most changes were towards hypo-methylation of tissues specific genes and oncogenic signature pathways, on the other hand during viral loss and development of KSHV-negative tumors changes are towards hyper-methylation. Mutational analysis of KSHV-infected cells and tumors revealed a set of mutations, including mutations in three inflammasome-related IFN response genes, that were absent in KSHV-infected cells but present in all KSHV-infected tumors in the same loci pointing to clonal selection \"in vivo\". This result suggests that in the context of in vivo tumorigenesis both these mutations and the virus may determine tumor growth. On the other hand, clustering analysis of mutations driving KSHV-negative tumors reveal a network comprising PDGFRA D842V, Pak1 and Nucleolin mutations implicated in cell proliferation. Our results have uncovered novel specific aspects of the interplay between host oncogenic alterations and virus-induced transcriptional effects as well as the epigenetic changes induced by KSHV infection and tumorigenesis. The existence virally-induced irreversible genetic and epigenetic oncogenic alterations support the possibility for hit-and-run KSHV sarcomagenesis which is consistent with pathological and clinical findings.\n\nAUTHOR SUMMARYWe performed whole genome RNA sequencing and CpG DNA methylation analysis in a mouse bone-marrow endothelial-lineage cells (mEC) transfected with the KSHVBac36 (mECK36 cells), that are able to form KSHV-infected tumors in nude mice, which were thoroughly characterized as KS-like tumors. This unique model allowed us to dissect genetic and epigenetic mechanisms of KSHV dependent and hit-and-run sarcomagenesis. We found that during KSHV in vivo lytic switch and KSHV-dependent tumorigenesis DNA methylation and Epigenetic regulation are among the most host-regulated pathways. CpG DNA methylation analysis during transformation supports the notion that loss of methylation (hypo-methylation) is the major epigenetic change during this process. Sequence analysis of KSHV-positive tumors revealed that KSHV tumorigenesis not only selects for the presence of the virus but also pre-existing host mutations that allow the KSHV oncovirus to express the oncogenic lytic program and creates a permissive environment of inflammation and viral tumorigenesis providing a selective advantage in vivo.

cancer biology↗