Investigation of viral and host determinants of gammaherpesvirus pathogenesis

Division Of Basic Sciences - Nci

Investigators

Linked publications & trials

Abstract

(1) STAT3 activation is associated with gammaherpesvirus latency and cancer in cell culture systems, but the target genes that drive latency, proliferation and transformation are not well-defined. As a prelude to studying the effects of STAT3 activation in KSHV itself, we used transgenic mice with a B cell-specific knock-out of STAT3 to determine that STAT3 signaling is critical for the establishment of gammaherpesvirus latency in primary B cells of the host. We also determined that the viral lytic gene transactivator RTA interacts with STAT3 in a cytokine-dependent manner. Our central hypothesis is that gammaherpesviruses require STAT3 to promote B cell latency. To define the STAT3-dependent gene expression program of infected B cells, we are performing RNAseq to compare the gene expression profile in infected cells with and without STAT3. Thus far we have identified a pro-viral role of STAT3 in dampening the interferon response in B cells via STAT3 inhibition of STAT1/2 and numerous antiviral interferon-stimulated genes. We will merge differentially expressed genes from all available gammaherpesvirus datasets to identify and prioritize common STAT3-regulated pathways and target genes. These will lead to further investigations of latency, reactivation, and transformation in KSHV and murine cell culture systems using conditional knock-out approaches. (2a) The numerous herpesvirus genes involved in nucleotide metabolism indicate that the nucleotide pool is a major restriction point in non-dividing cells. We recently reported that the enzymatic properties of the viral uracil DNA glycosylase (UNG) synergize with the viral nucleotide metabolism protein dUTPase to promote pathogenesis and counter recombination-based deletions in the viral genome. We are currently exploring protein binding partners of the viral UNG. In collaboration with Kevin McBride, we are examining the differential biochemical properties of the viral and host UNG. (2b) Also, in collaboration with the McBride lab, we analyzed the immunoglobulin repertoire of infected and uninfected B cells from infected mice using next generation sequencing. This analysis revealed that gamamherpesvirus-infected cells undergo clonal expansion yet only a few clones were shared between the infected and uninfected germinal center B cells. There is also evidence for receptor editing and clear bias for specific IghV genes in the infected B cells. These novel data indicate the gammaherpesviruses occupy a distinct niche in the infected host and takes an active role in subverting the immunoglobulin repertoire in the B cells that it infects. This supports the existence of a critical strategy that is shared with KSHV and EBV, namely that these viruses bypass normal selection processes, placing the B cells at risk for genetic instability and for producing immunoglobulins with pathologic potential. I am also collaborating with other NCI investigators and clinicians in HAMB to analyze the B cell repertoire of KSHV-infected cells in HIV-associated primary effusion lymphoma and lymph node material of HIV-infected KSHV-multicentric Castleman disease patients enrolled in HAMB clinical trials. This will enable us to examine the clonality and source of the B cell transformation. Importantly, this might inform the possible etiology of these diseases which often occur in a concurrent or sequential manner in people living with HIV. (3) We are examining the mechanism by which the viral ribonucleotide reductase large subunit relocalizes host nuclear defense factors, termed PML-NB into track-like structures. We hypothesize that this relocalization by the virus neutralizes their antiviral functions and promotes productive infection. (4) With regard to the development of direct animal models of Kaposi sarcoma, a hallmark tumor of AIDS, I am collaborating with clinicians in the HIV and AIDS Malignancy Branch and in the Center for Advanced Preclinical Research (CAPR) of NCI Frederick in more translational projects towards the establishment of a patient-derived xenograft model. KS biopsy material from HIV infected patients in the HAMB clinical cohorts is being implanted into immunodeficient animals. Thus far, we observe KSHV-infected endothelial cell proliferation and expansion in the xenografts. Cell lines derived from this system that maintain KSHV infection and drive tumors in mice will be a tremendous advancement for the field since there is no way to maintain KSHV+ endothelial cells upon explant and there is no pre-clinical animal model of Kaposi sarcoma to screen for effective drug interventions of this AIDS-defining cancer.