Emerging retrovirus meets match in emerging retrovirologist
by Alex Compton
Retroviruses, notorious for causing immunodeficiency in primates, cats and cows, were first studied by cancer biologists to understand mechanisms of cell transformation and carcinogenesis. In fact, the first retrovirus discovered was an agent responsible for neoplasia in chickens. Tumor-inducing retroviruses were soon revealed in mammalian species as well, effectively bridging the fields of retrovirology and cancer biology for decades to come.
Beginning in 2006, scientists and the public alike were reminded of this relationship when Xenotropic Murine Leukemia Virus-Like Retrovirus (XMRV) was implicated in two human diseases with unknown etiology: prostate cancer and chronic fatigue syndrome. These disease associations, based primarily on detection of viral DNA in patient cells and laboratory cell lines, invited considerable scrutiny from virologists seeking to establish whether this virus represented a veritable human pathogen.
Dr. Dusty Miller is a central figure in the study of oncogenic retroviruses at the Fred Hutchinson Cancer Research Center, which holds a rich history of incorporating infectious disease research into the study of cancer. Serendipitously, he was swept into the XMRV debate when the virus turned up in a cell line at the Hutch. Dr. Muneesh Tewari and MCB graduate student Emily Knouf used electron microscopy to reveal that a prostate carcinoma cell line known as 22Rv1 was pumping out sinister black particles into the culture medium. Miller and his lab members genotyped the virus and found that it was nearly identical to XMRV. Wielding the tools and expertise to characterize the virus further, Miller was poised to answer fundamental questions about how XMRV infects human prostate epithelial cells and causes disease.
Miller’s initial work showed that the retrovirus did not transform cells in culture, suggesting that it may not play a direct role in oncogenesis. However, his occupation with XMRV did not end there. He enlisted Andy Vaughan, an MCB graduate student at the time, to study interactions between the virus and the human membrane protein Xpr1. Apart from its role as receptor for XMRV, very little was known about the normal cellular function of Xpr1. However, sequence homology to a yeast protein hinted that it may function as a G-protein coupled receptor. Vaughan performed immunoprecipitation experiments to demonstrate that Xpr1 binds the Gβ subunit, implicating the receptor in G-protein mediated pathways that signal through the secondary messenger cyclic AMP (cAMP). “As it turns out, by overexpressing Xpr1, cyclic AMP levels increase substantially inside the cells,” Vaughan said, suggesting that the receptor normally functions as a positive regulator of intracellular cAMP. When he infected cells with the virus, cAMP levels plummeted. “Our model is that the viral envelope is binding Xpr1 and disrupting its normal function,” he said. He wanted “to characterize the downstream effectors of Xpr1 and try to get a better idea of how certain signaling networks are being affected by this.” Interestingly, Vaughan found that XMRV infection triggered programmed cell death in a neuroblastoma cell line, whereas cell lines derived from other tissues fared much better. This led him to hypothesize that the engagement of XMRV to Xpr1 causes neuronal cytopathicity by virtue of downregulating cAMP. “In this particular cell type, there is direct evidence that cAMP levels and G-protein signaling are really important for their survival,” he said.
While wrapping up this project and scheduling his dissertation defense, a flurry of papers examining the ties between XMRV, prostate cancer, and chronic fatigue syndrome came up empty-handed. Many labs independently showed that XMRV detection in human samples was simply a PCR artifact—owing to the ubiquity of cancer cell lines like 22Rv1 in laboratories around the globe, XMRV is a common laboratory contaminant. The debate was finally put to rest when Miller, Vaughan, and Ramon Mendoza found that a portion of the XMRV genetic sequence was identical to that of an endogenous retrovirus found in mouse cells, which suggested that the virus originated in mice. Around the same time, the laboratories of John Coffin at Tufts University and Vinay Pathak at the National Cancer Institute discovered that XMRV was generated by recombination of two mouse retroviruses during the serial passage of human tumor tissue in laboratory mice.
Although a causative role for XMRV in human disease is now untenable, the work of Vaughan and Miller is a reminder of how retroviruses continue to yield insight into the biology of the hosts they infect. Their work reveals that Xpr1 is a novel G-protein coupled receptor whose activity is especially important for neuron survival, and that a breach in Xpr1-mediated signaling may play a role in mechanisms of neurologic disease.
Vaughan, who defended his dissertation in May 2011, is now a postdoctoral scholar at the University of California-San Francisco.