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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 18 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, and typically inhibit their translation. The key to understanding the function of the latent viral miRNAs is to identify their viral and host mRNA targets. However, the short length and imperfect homology of the targeted region makes bioinformatic prediction of targets extremely challenging. We have developed a variety of experimental approaches to this problem. Using these approaches, we have identified a number of miRNA targets in both the host and viral transcriptomes. One early theme that has emerged from this work is that the KSHV miRNAs can modulate the stability of the latent state, both positively and negatively. This provides a finer level of control than is possible with transcriptional regulation alone, and helps to assure both the stability and reversibility of latency.
As noted above, a prominent feature of KS is the presence of inflammation in and around the tumor. Clinical and pathologic evidence suggests that this inflammatory state plays an important role in the progression of KS lesions. 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, was originally discovered in our laboratory; it 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.
In parallel with studies of host transcription during the lytic cycle, we have recently been applying contemporary genomic methodologies to analyze the lytic viral transcriptome in greater detail. This has led to the recognition that, like its host counterpart, the viral transcriptome includes numerous large, stable noncoding RNAs—many of which are antisense to important viral genes. One of these is a 10-kb RNA complementary to at least 7 of the viral pre-miRNAs as well as to several key latent coding regions. This suggests the possibility of previously unrecognized forms of regulation of these products—a lead we are now pursuing experimentally.
Because progression of KSHV infection in vivo requires escape from host immune responses, we have screened lytic-cycle genes for modulators of such responses. By screening for genes that block the ability of interferon (IFN) to trigger the induction of IFN-responsive genes, we have identified a gene (designated RIF for regulator of interferon function) that blocks IFN signaling very proximal to the IFN receptor, thereby preventing activation of the downstream transcription activators known as STAT-1 and STAT-2. A different screen for regulators of innate immune responses has led us to a second viral gene. This locus encodes a distant relative of RIF and acts to down-regulate surface IFN receptors, as well as a variety of other cytokine receptors. These functions of this gene appear to have been unique evolutionary inventions of KSHV—the gene's homologs in other herpesviruses lack these immunoregulatory activities.
Finally, because KSHV infects B cells as well as endothelial cells in vivo, we have used primary tonsillar lymphoid cultures to develop a cell culture system for B cell infection. Infection in such B cells behaves differently from infection in nonlymphoid cells, with high rates of spontaneous lytic reactivation. This reactivation is subject to control by primary T cells, and the nature of this control appears to be novel. It requires T cell activation and cell-cell contact but is not MHC (major histocompatibility complex)-restricted, is not directed at virus-specific antigens, and does not operate via target cell killing. We are investigating the cellular and molecular mechanisms underlying this activity.
Searching for New Viral Pathogens in Human Disease
In collaboration with Joseph DeRisi (HHMI, University of California, San Francisco), we have also undertaken major efforts to identify previously unrecognized viruses in a variety of human 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. We also employ high-throughput cDNA sequencing as an independent approach, where indicated.
With these methods, we have examined a large number of specimens from patients with a variety of acute and chronic diseases. In a study of more than 200 cases of respiratory infection, 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 new clade of rhinoviruses that are very divergent from those previously known to exist. From a patient with an acute influenza-like syndrome, we identified a human cardiovirus distantly related to murine viruses that produce encephalitis and myocarditis in mice. Our epidemiologic studies show that infection by this agent (HTCV) is rare in respiratory disease but is found in approximately 1 percent of diarrheal stools. Serologic screens indicte that HTCV infection is widespread in human populations. Further clinical studies are needed to determine if this new virus can (like its murine relative) be implicated in central nervous system and cardiac infection.
In collaboration with Robert Silverman and Eric Klein (Cleveland Clinic), we have 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 γretrovirus 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. Our findings represent the first definitive evidence that γretrovirus infections 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.
Recently we have discovered a new bornavirus in an epidemic gastrointestinal disease of exotic parrots. This avian disease is not only of veterinary importance but is also clinically and pathologically similar to a human disorder known as achalasia. In achalasia, inflammation along enteric nerves leads to denervation of the esophagus, resulting in disordered swallowing and malnutrition. The cause of human achalasia has long been a mystery, with autoimmunity and viral infection (or some combination of the two) being the leading proposals. Our findings in birds provide the first direct evidence that virus infection can trigger achalasia-like pathophysiology, and have led us to an ongoing examination of human achalasia specimens in search of an etiologic agent in that enigmatic human disease.
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 January 20, 2010
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