The goals of this investigation are to explore and define the relationship of the cardiovirus genus to other members of the picornavirus family and to exploit the unique features of cardioviruses to examine fundamental molecular questions about picornavirus translation, proteolytic processing, morphogenesis and host interaction. The RNA picornaviruses are one of the best understood and most thoroughly accessible experimental systems in all of biology. Natural infections with cardioviruses, like encephalomyocarditis virus (EMCV), kill nearly every cell in the brain, pancreas and heart of a mouse, within 3 days. The virus does this with apparent impunity to cellular antiviral defenses by subverting innate immunity traps and crippling the capacity-of an infected cell to mount a defense or trigger an alarm. The molecular battleground inside infected cells pits viral protease 3Cpro, and two unique cardiovirus proteins, L and 2A, enzymes honed by evolution for their special anti-cellular purposes, against the complete array of innate host defenses. The outcome rarely varies. Within 2-3 hours of infection EMCV brings to a halt cellular mRNA transcription, cap-dependent mRNA translation, antiviral signal transduction, and active protein/RNA exchange between the nucleus and cytoplasm. The virus replicates with fecundity and the cell dies before it ever triggers an alarm. At the ultimate molecular level, the activities of these proteins instigate the cascade of events that set off or prevent an episode of disease. The next phase of this project will examine the biochemistry and molecular pathways of EMCV L (Leader), the first viral (or cellular) protein known to bind and inactivate Ran GTPase cycling, the crucial, ubiquitous regulatory system for all protein and nucleic acid trafficking into and out of the nucleus. The project also extends studies on EMCV 2A, a protein that subverts nucleoli and converts normal ribosomes into configurations which prevent translation of host mRNAs, and it continues examinations of 3Cpro, an enzyme whose precursors have anti-host proclivities that go far beyond normal processing of the viral polyprotein. These objectives build directly upon experimental foundations developed during the preceding 26 years of the program.
The specific aims of are: (1) To resolve the NMR structure of Mengo L (Leader) protein as it interacts with Ran GTPase. (2) To characterize the biochemistry of cardiovirus L:Ran interactions which inhibit RanGDP/GTP cycling in cell-free extracts. (3) To identify within cells, the nucleocytoplasmic trafficking steps abrogated by cardiovirus L protein. (4) To define molecular advantages to cardioviruses, for encoding a potent inhibitor of Ran. (5) To clarify the respective roles of cardioviral L, 2A and 3C, in pol-2 transcriptional shutoff within the nuclei of infected cells. Relevance to Public Health: It is rare to establish productive viral infections that lead to disease if the first infected host cells or host immune system are able to fight back effectively. In a war won by stealth, an inadvertent triggering of intracellular or extracellular immunological alarms usually spells disaster (and clearance) for the virus. It's the job of the first viral proteins produces in the first 2-3 hrs of infection, to shutoff essential host response systems. This project examines the molecular pathways for how this happens with RNA picornavi ruses in the cardiovirus genus.
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