The hypothesis that 26S (interjacent) RNA made in cedls infected by group A arboviruses serves as messenger RNA will be examined by hybridization methods using Sindbis virus. Since only a limited spectrum of virus-specific proteins constitute the bulk of protein synthesis in infected cells, only about one half of the base sequences found in virion RNA may predominate in the interjacent RNA used as messenger. Thus the interjacent and virion RNA should exhibit different hybridization kinetics in tests with labeled replicative form RNA purified from infected chick embryo cells. Both the bulk interjacent RNA and the virus-specific RNA from subcellular fractions active in protein synthesis will be used in the hybridization test. Competitive hybridization will also be used to determine degree of RNA homology between Sindbis virus and other closely and more distantly related group A arboviruses and between Sindbis virus and a group B arbovirus, Japanese encephalitis virus. A series of temperature sensitive mutants of Japanese encephalitis virus will be isolated and categorized genetically by complementation. The mutants will be tested for ability to synthesize virad RNA at the nonpermissive temperature and then will be further characterized by various biochemical tests. In an effort to reproduce the intracellular morphogenesis of Sindbis virus, suitable conditions for in vitro nucleocapsid assembly and envelopment will be sought. Capsid protein for the assembly of nucleocapsids will be prepared by controlled degradation of nucleocapsids purified from infected cells. Reassembly, with and without added viral RNA, will be assessed by gradient centrifugation and electron microscopy. Envelopment of nucleocapsids will be sought in mixtures of plasma membranes from infected cells and genetically distinct nucleocapsids. Shifts in ionic strengths will be used to promote in vitro envelopment.