THE EMERGENCE OF CHIMERIC RNA-DNA VIRUSES. VIRUSES ARE PROBABLY AS OLD AS LIFE ITSELF AND HAVE BEEN INVOLVED IN MOST MAJOR EVOLUTIONARY TRANSITIONS. CURRENTLY THERE ARE THREE MAIN GROUPS OF KNOWN VIRUSES THOSE THAT HAVE RNA GENOMES THOSE THAT HAVE DNA GENOMES AND THE RETROVIRUSES THAT USE CELLULAR DNA INTERMEDIATES TO REPLICATE THEIR RNA GENOMES. UNTIL VERY RECENTLY IT WAS THOUGHT THAT VIRUSES DID NOT EXCHANGE GENETIC INFORMATION BETWEEN VIRUSES OF DIFFERENT GROUPS. HOWEVER IN 2012 IN BOILING SPRINGS LAKE (BSL) WE DISCOVERED THE FIRST EXAMPLE OF A HERETOFORE UNDISCOVERED WIDESPREAD GROUP OF VIRUSES WITH GENOMES THAT APPARENTLY AROSE BY AN UNPRECEDENTED RNA-DNA RECOMBINATION EVENT BETWEEN 2 COMPLETELY DIFFERENT CLASSES OF VIRUSES THE CHIMERIC VIRUSES (CHIV). THE FREQUENCY AND MECHANISM OF THE RECOMBINATION GENERATING CHIVS ARE UNCLEAR BUT CRITICAL FOR UNDERSTANDING VIRUS EVOLUTION THE EMERGENCE OF NEW VIRUSES AND POSSIBLY ALLOW INSIGHTS INTO THE TRANSITION BETWEEN RNA AND DNA IN THE EVOLUTION OF CELLULAR LIFE ON EARTH. WE WILL ADDRESS THE FREQUENCY AND MECHANISM OF EMERGENCE OF CHIV FORMATION BY PURSUING 3 SPECIFIC AIMS: 1. ISOLATE AND CHARACTERIZE THE CHIV REPLICATION-INITIATION PROTEIN IMPLICATED IN THE RNA-DNA RECOMBINATION. 2. ISOLATE AND CHARACTERIZE THE CHIV CAPSID PROTEIN A RNA-VIRUS-LIKE CAPSID PROTEIN THAT ENCAPSIDATES DNA. 3. DETERMINE THE CHIV HOST BY SCREENING ENVIRONMENTS KNOWN TO HARBOR CHIVS USING PACS PHAGE-FISH AND FACS. AIM 1: WE WILL CLONE EXPRESS AND PURIFY CHIV REP ENDONUCLEASE-LIGASE PROTEINS WHICH ARE LIKELY TO HAVE THE ENZYMATIC ACTIVITY LINKING DNA AND RNA. PROTEINS WILL BE EXPRESSED AND PURIFIED USING STANDARD RECOMBINANT DNA AND PROTEIN PURIFICATION TECHNIQUES. ACTIVITY WILL BE TESTED USING SENSITIVE DNA AND RNA LIGATION ASSAYS. THESE RESULTS WILL ADDRESS THE MECHANISM AND FREQUENCY OF RECOMBINATION IN VITRO. AIM 2: WE WILL CLONE EXPRESS AND PURIFY THE CHIV CAPSID PROTEIN (CP). THE CAPSID PROTEIN IS HIGHLY CONSERVED IN ALL CHIVS AND IS CLEARLY MONOPHYLETIC WITH TOMBUSVIRUS RNA VIRUS CAPSID PROTEINS. HOWEVER CHIVS CPS PACKAGE DNA. PURIFIED CHIV CP (METHODS AS IN AIM 1) WILL BE TESTED USING BIOPHYSICAL TOOLS INCLUDING ANALYTICAL ULTRA-CENTRIFUGATION SPECTROSCOPY SMALL ANGLE NEUTRON SCATTERING AND ELECTRON MICROSCOPY. ANTIBODIES WILL BE PRODUCED FOR FACS. THESE RESULTS WILL ASSESS HOW STRAIGHTFORWARD IT IS TO CHANGE THE SPECIFICITY OF A VIRUS CP AND PROVIDE INSIGHTS INTO THE FREQUENCY OF EMERGENCE OF CHIVS. AIM 3: TO DATE NO CHIV VIRUS OR HOST HAS BEEN ISOLATED. THE LARGEST DIVERSITY OF CHIV SEQUENCES FOUND TO DATE IS IN FRESHWATER LAKES SAMPLES. WE WILL SCREEN LOW-DIVERSITY SAMPLES WITH PCR-ACTIVATED CELL SORTING (PACS) A CUTTING-EDGE EXTREMELY HIGH THROUGHPUT METHOD FOR SEPARATION OF VIRUS SEQUENCE ASSOCIATED GENOMES VIRUSES AND HOSTS FOR POTENTIAL CHIV HOSTS. WE WILL ALSO USE PHAGE-FISH AND FLOW-CYTOMETRY (SEE AIM 2) ON THESE SAMPLES. WE WILL ALSO USE BIOINFORMATICS TOOLS TO TRY TO IDENTIFY POTENTIAL HOSTS. WE WILL ALSO SCREEN LOCAL FRESHWATER LAKES FOR CHIV GENOMES VIRIONS AND HOSTS. WHEN A HOST IS IDENTIFIED WE WILL ATTEMPT GROWTH INFECTION AND VIRUS CHARACTERIZATION. THESE RESULTS WILL ALSO ADDRESS THE FREQUENCY OF CHIV FORMATION. NASA RELEVANCE: THIS RESEARCH SEEKS TO FURTHER OUR FUNDAMENTAL UNDERSTANDING OF EVOLUTION AND THE BASIC BIOCHEMICAL PROCESSES THAT DRIVE IT. THIS RESEARCH WILL CHARACTERIZE NOVEL BIOCHEMICAL CATALYTIC FUNCTIONS OF PROTEINS. THERE IS A DISTINCT POSSIBILITY THAT CHIVS OR A SIMILAR ACTIVITY PLAYED A ROLE IN THE TRANSITION FROM THE RNA WORLD TO DNA-WORLD . THESE UNKNOWN BIOCHEMICAL MECHANISMS HAVE IMPORTANT IMPLICATIONS FOR THE EVOLUTION OF BOTH PRE-BIOTIC AND ADVANCED LIFE MAKING THIS PROPOSED WORK DIRECTLY RELEVANT TO TWO AREAS OF EMPHASIS WITHIN THE EXOBIOLOGY PROGRAM. FURTHERMORE UNDERSTANDING THE EMERGENCE OF NEW VIRUSES IS VERY IMPORTANT FOR MANNED SPACE FLIGHT AND LIFE SUPPORT.