Identification of attenuating mutations on the reovirus type 3 S1 double-stranded RNA segment with a rapid sequencing technique.

Reovirus type3 variants withmutations inthemajorneutralization domainofthe urlprotein have attenuated neurovirulence andrestricted neurotropism. We devised a variation oftherapid RNA sequencing technique tofacilitate theanalysis ofdouble-stranded RNA. We sequenced theS1double-stranded RNA segment, whichencodes theirl protein, offive attenuated reovirus type3variants. Fourofthevariants have changes incodon419,anda fifth variant hasa changeatcodon340,allofwhichresulted inaminoacid substitutions inthecxlprotein. We identified twosites on thereovirus type3crlprotein thatplay acritical role inneurovirulence. We usedthemammalian reoviruses asamodeltobetter understand viral pathogenesis andtodefine atamolecular level thedeterminants responsible forthecapacity ofthese viruses toinfect specific cells andtissues (tropism). The reovirus genomeconsists of10segments ofdouble-stranded (ds) RNA (reviewed inreference 8).Infection ofneonatal micewithmammalian reovirus type3 produces a fatal encephalitis secondary tolocalization ofreovirus type3in neurons (10, 13). Thereovirus S1ds-RNAsegment encoding theclprotein (viral hemagglutinin) determines thetropism ofreovirus forneurons (21, 23,24)andpituitary cells (12). Theo1protein isalsoresponsible fordetermining anumber ofvirus-host cellinteractions, including serotype-specific humoralandcellular immunity, binding tocellular microtubules, andinhibition ofcellular DNA syntheis (8). To identify different domains ofthereovirus type3hemagglutinin, we isolated ol-specific monoclonal antibodies (3). Analysis ofthecrlprotein byusing these monoclonal antibodies indicated that there areatleast three distinct domains (20). Onedomain, termedtheneutralization domain, is primarily defined bytwomonoclonal antibodies, A2andG5, thatshowhightiters ofneutralizing activity. TheG5monoclonal antibody wasusedtogenerate viral variants (designatedA,F,andK)thatresist neutralization (19). These variant viruses aremarkedly less virulent thantheparental reovirus type3virus fromwhichtheywereselected. In addition, examination ofbrain sections ofneonatal mice injected intracerebrally withthevariants showsthatthey causetissue injury inrestricted regions ofthemousebrain (18). Theseresults indicate thatmutations inthereovirus type3clprotein causemarkedalterations inviral virulence andtropism. Inaddition totheoriginal attenuated reovirus type3 variants, twomorereovirus type3 viruses with antigenically altered olproteins wereselected aspreviously described (19), (designated A14andA17), except thatthe parental reovirus type3 wasgrowninthepresence of neutralizing monoclonal antibody A2instead ofG5.Thus,