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The Biology of Human Viral Pathogens
Summary: Don Ganem is interested in identifying human viral pathogens and in determining the mechanisms by which they replicate in their hosts and cause disease.
Our laboratory studies the molecular mechanisms by which pathogenic viruses infect the human host and cause disease. Our work centers around two main areas: (1) the pathogenesis of Kaposi's sarcoma (KS), a tumor linked to infection with a distinctive human herpesvirus, and (2) the discovery of novel viral pathogens associated with acute and chronic human diseases.
Kaposi's Sarcoma and Its Causative Herpesvirus
KS is a distinctive tumor characterized by the deregulated growth of endothelial cells (cells that normally line blood vessels). In addition to proliferation, KS is characterized by a strong association with local inflammation and abnormal angiogenesis. KS pathogenesis requires infection by a virus called KS-associated herpesvirus (KSHV). Like all herpesviruses, KSHV is capable of producing either a latent infection, in which only a handful of viral genes are expressed, or a lytic infection, in which full viral gene expression leads to cell death and the release of infectious virus particles. Most KS tumor cells display latent infection, but 1–2 percent of the cells in the lesion are in the lytic cycle.
Because of the prominence of latent infection in KS, we have devoted considerable effort to understanding the KSHV latency program and its links to cell survival, angiogenesis, and proinflammatory signaling. We and others have shown that KSHV latency entails the production of up to seven virus-encoded proteins and 17 small noncoding RNAs known as microRNAs (miRNAs).
The KSHV miRNAs are produced from latent viral mRNAs derived from a locus encoding the viral kaposin proteins (see below). MicroRNAs base-pair to short sequences in the 3'-noncoding regions of messenger RNAs. They typically inhibit the translation of mRNAs; this inhibition is often accompanied by modest changes in mRNA abundance. The key to understanding the function of the viral latent miRNAs is to identify their mRNA targets. However, the short length and imperfect homology of the targeted region makes bioinformatic prediction of targets extremely challenging. Nonetheless, by combining computational approaches with expression profiling under several conditions we have recently made considerable headway in identifying host mRNAs targeted by KSHV. Several of these host mRNAs function in the regulation of cell survival and angiogenesis.
As noted above, a prominent feature of KS is the presence of inflammation in and around the tumor. Our efforts to understand the link(s) between KSHV infection and inflammatory signaling have led us to two latent proteins. One of these, kaposin B, which was originally discovered in our laboratory, is an activator of the proinflammatory p38 MAP kinase signaling cascade. Both p38 and its downstream target MK2 are activated, triggering a stabilization of AU-rich element (ARE)-containing mRNAs. Many ARE-bearing mRNAs encode cytokines and growth factors, including several implicated in KSHV-related disease states (e.g., IL-6, VEGF). Kaposin B is thus a strong candidate for an important pathogenetic role in KS.
The second important KSHV latency gene encodes the v-FLIP protein, which strongly activates the transcription factor NFκB. This results in transcriptional up-regulation of many proinflammatory genes, including cytokines and chemokines also known to promote the survival and proliferation of KS spindle cells in vitro. In addition to its proinflammatory effects, NFκB activation in endothelial cells also induces a dramatic cytoskeletal rearrangement that gives the cell a spindle shape—the morphologic signature of KS tumor cells in vivo.
The lytic cycle of KSHV also contributes importantly to KS development, largely via paracrine signaling. We continue to examine the effects of lytic KSHV infection on host gene expression, including the expression of both mRNAs and miRNAs. In general, host gene expression is strongly impaired by KSHV replication, via activation of host mRNA turnover. However, 2 percent of host transcripts escape this degradation and are up-regulated. This includes several proinflammatory cytokines (e.g., IL-6 and CXCL2), as well as known regulators of angiogenesis and endothelial cell function. The formidable task now before us is to explicitly evaluate the contributions of each such molecule to KS pathogenesis.
Finally, we have initiated a search among lytic-cycle genes for viral modulators of host immune responsiveness. By screening for genes that block the ability of interferon (IFN) to trigger the induction of IFN-responsive genes, we have identified two viral genes that can thwart this important arm of innate antiviral defense. One of these has previously been found to have another role in the viral replicative cycle, while the second encodes an entirely novel gene product. Work with the latter protein shows that its expression blocks IFN signaling very proximal to the IFN receptor, thereby preventing activation of the downstream transcription activators known as STAT-1 and STAT-2.
Searching for New Viral Pathogens in Human Disease
In collaboration with Joseph DeRisi (HHMI, University of California, San Francisco), we have also begun major efforts to identify novel human viral pathogens in a variety of disease states. Our approach employs a DNA microarray that bears the most conserved sequences of all known viruses of humans, animals, plants, and microbes. RNA extracted from clinical specimens is amplified, labeled, and hybridized to the array, and the hybridization patterns are inspected. Although the method clearly is biased toward the identification of viral genomes that are related to known virus families, it can detect novel agents whose sequences are quite divergent from existing isolates.
With this method, we have examined a large number of specimens from patients with a variety of acute and chronic diseases. In a study of more than 80 cases of respiratory infection in asthmatic hosts, we identified the likely pathogen in 65 percent of cases—a clear improvement over standard viral culture or antigen assays. This led to recognition of a novel clade of rhinoviruses that are very divergent from those previously known to exist. In addition, we found that the coronaviruses now circulating in our population do not correspond to those usually listed in textbooks of medicine. Rather, they represent newly identified viruses whose epidemiology is only starting to become clear. Similar findings have been made in a large cohort of pediatric patients with acute respiratory infection.
In collaboration with Robert Silverman and Eric Klein (Cleveland Clinic), we have also studied men with familial prostate cancer putatively linked to mutations in RNase L (an important mediator of IFN's antiviral effects). We identified a novel γ-retrovirus in tissue from 40 percent of men bearing the RNase L R462Q mutation; this agent was present in <2 percent of men with wild-type RNase L. This virus, termed XMRV, is closely related to xenotropic retroviruses of mice. Xenotropic viruses were discovered first in mice, but replicate preferentially in nonmurine cells; it has long been wondered if such viruses can produce human infection in vivo. Our findings represent the first definitive evidence that such infections indeed exist in man. The virus is not found in the tumor cells, but resides in a small subset of stromal cells in the prostate. Therefore, XMRV is not a traditional oncogenic retrovirus, and its relationship to prostate cancer is uncertain. The clear linkage of infection to deficiencies in RNase L is, however, the first strong evidence in humans that this enzyme is important in antiretroviral defense.
Very recently, we identified a novel picornavirus in a patient with an acute influenza-like syndrome; this virus is distantly related to murine viruses that produce encephalitis and myocarditis in mice, and we have initiated epidemiologic studies to determine if this new virus can be implicated in human central nervous system and cardiac infection.
The National Institutes of Health provided support for some of our studies of KSHV, and the use of the viral array for respiratory and other infections is also supported by the Doris Duke Charitable Foundation.
Last updated: July 17, 2007
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