Reverse genetics system and fucntion of NSM protein of Rift Valley fever virus (family Bunyaviridae, genus phlebovirus)

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dc.contributor.advisorShinji Makinoen_US
dc.contributor.committeeMemberThomas K. Hughesen_US
dc.contributor.committeeMemberSankar Mitraen_US
dc.contributor.committeeMemberRobert E. Johnstonen_US
dc.creatorSungyong Wonen_US
dc.date.accessioned2011-12-20T16:05:31Z
dc.date.available2008-06-17en_US
dc.date.available2011-12-20T16:05:31Z
dc.date.created2007-11-13en_US
dc.date.issued2007-11-07en_US
dc.description.abstractRift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and results in large epidemics among humans and livestock. Furthermore, it has potential as a bioterrorist agent and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. I established a T7 RNA polymerase-based reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phleboviruses. Using this system, RVFV carrying substitution mutations of the M gene preglycoprotein region (pre-Gn region), one lacking NSm protein expression, one lacking 78-kDa protein expression, and one lacking expression of both proteins, were rescued and compared in cell culture. All of the mutants and their parent virus produced plaques with similar sizes and morphologies in Vero E6 cells and had similar growth kinetics in Vero, C6/36, and MRC5 cells, demonstrating that the NSm and 78-kDa proteins were not needed for the virus to replicate efficiently in cell culture. A competition-propagation assay revealed that the parental virus was slightly more fit than the mutant virus lacking expression of both proteins. To determine the biological functions of the NSm and 78-kDa proteins, I generated the mutant virus, arMP-12-del21/384, carrying a large deletion in the pre-Gn region of the M segment. Neither NSm nor the 78-kDa proteins were synthesized in arMP-12-del21/384-infected cells. Although arMP-del21/384 and its parental arMP-12 showed similar virus growth kinetics, viral RNA, and proteins accumulation in infected cells, arMP-12-del21/384 infection induced extensive cell death and produced larger plaques than did the arMP-12 infection. arMP-12-del21/384 replication triggered apoptosis, including caspase-3 activation, cleavage of its downstream substrate, poly-ADP-ribose polymerase, and activation of initiator caspases, caspase-8 and -9, early in infection as compared with arMP-12 replication. NSm expression in arMP-12-del21/384-infected cells suppressed the severity of caspase-3 activation. Further, NSm protein expression inhibited staurosporine-induced activation of caspase-8 and -9, demonstrating that other viral proteins were dispensable for the NSm’s function in inhibiting apoptosis. RVFV NSm protein is the first identified Phleobovirus protein that has antiapoptotic function.en_US
dc.format.mediumelectronicen_US
dc.identifier.otheretd-11132007-104341en_US
dc.identifier.urihttp://hdl.handle.net/2152.3/253
dc.language.isoengen_US
dc.rightsCopyright © is held by the author. Presentation of this material on the TDL web site by The University of Texas Medical Branch at Galveston was made possible under a limited license grant from the author who has retained all copyrights in the works.en_US
dc.subjectRift Valley fever virusen_US
dc.subjectReverse genetics systemen_US
dc.subjectPhlebovirusen_US
dc.subjectNSm proteinen_US
dc.subjectBunyavirusen_US
dc.subjectapoptosisen_US
dc.subject78-kDa proteinen_US
dc.titleReverse genetics system and fucntion of NSM protein of Rift Valley fever virus (family Bunyaviridae, genus phlebovirus)en_US
dc.type.genredissertationen_US
dc.type.materialtexten_US
thesis.degree.departmentMicrobiology and Immunologyen_US
thesis.degree.grantorThe University of Texas Medical Branchen_US
thesis.degree.levelDoctoralen_US
thesis.degree.namePhDen_US

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