The genomes of many positive stranded RNA viruses and of allretroviruses are translated as large polyproteins which are proteolyticallyprocessed by cellular and viral proteases. Viral proteases are structurallyrelated to two families of cellular proteases, the pepsin-like and trypsin-likeproteases. This thesis describes the proteolytic processing of severalnonstructural proteins of dengue 2 virus, a representative member of theFlaviviridae, and describes methods for transcribing full-length genomicRNA of dengue 2 virus. Chapter 1 describes the in vitro processing of thenonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a systemthat allows identification of residues within the protease that are directly orindirectly involved with substrate recognition. Chapter 3 describesmethods to produce genome length dengue 2 RNA from cDNA templates.
The nonstructural protein NS3 is structurally related to viral trypsinlikeproteases from the alpha-, picorna-, poty-, and pestiviruses. Thehypothesis that the flavivirus nonstructural protein NS3 is a viralproteinase that generates the termini of several nonstructural proteins wastested using an efficient in vitro expression system and antisera specific forthe nonstructural proteins NS2B and NS3. A series of cDNA constructswas transcribed using T7 RNA polymerase and the RNA translated inreticulocyte lysates. Proteolytic processing occurred in vitro to generateNS2B and NS3. The amino termini of NS2B and NS3 produced in vitrowere found to be the same as the termini of NS2B and NS3 isolated frominfected cells. Deletion analysis of cDNA constructs localized the proteasedomain necessary and sufficient for correct cleavage to the first 184 aminoacids of NS3. Kinetic analysis of processing events in vitro and experimentsto examine the sensitivity of processing to dilution suggested that anintramolecular cleavage between NS2A and NS2B preceded anintramolecular cleavage between NS2B and NS3. The data from theseexpression experiments confirm that NS3 is the viral proteinaseresponsible for cleavage events generating the amino termini of NS2B andNS3 and presumably for cleavages generating the termini of NS4A and NS5as well.
Biochemical and genetic experiments using viral proteinases havedefined the sequence requirements for cleavage site recognition, but havenot identified residues within proteinases that interact with substrates. Abiochemical assay was developed that could identify residues which wereimportant for substrate recognition. Chimeric proteases between yellowfever and dengue 2 were constructed that allowed mapping of regionsinvolved in substrate recognition, and site directed mutagenesis was usedto modulate processing efficiency.
Expression in vitro revealed that the dengue protease domainefficiently processes the yellow fever polyprotein between NS2A and NS2Band between NS2B and NS3, but that the reciprocal construct is inactive.The dengue protease processes yellow fever cleavage sites more efficientlythan dengue cleavage sites, suggesting that suboptimal cleavage efficiencymay be used to increase levels of processing intermediates in vivo. Bymutagenizing the putative substrate binding pocket it was possible tochange the substrate specificity of the yellow fever protease; changing aminimum of three amino acids in the yellow fever protease enabled it torecognize dengue cleavage sites. This system allows identification ofresidues which are directly or indirectly involved with enzyme-substrateinteraction, does not require a crystal structure, and can define thesubstrate preferences of individual members of a viral proteinase family.
Full-length cDNA clones, from which infectious RNA can betranscribed, have been developed for a number of positive strand RNAviruses, including the flavivirus type virus, yellow fever. The technologynecessary to transcribe genomic RNA of dengue 2 virus was developed inorder to better understand the molecular biology of the dengue subgroup. A5' structural region clone was engineered to transcribe authentic dengueRNA that contains an additional 1 or 2 residues at the 5' end. A 3'nonstructural region clone was engineered to allow production of run offtranscripts, and to allow directional ligation with the 5' structural regionclone. In vitro ligation and transcription produces full-length genomicRNA which is noninfectious when transfected into mammalian tissueculture cells. Alternative methods for constructing cDNA clones andrecovering live dengue virus are discussed.
