Proteolytic processing of the nonstructural proteins of dengue 2 virus

The genomes of many positive stranded RNA viruses and of all retroviruses are translated as large polyproteins which are proteolytically processed by cellular and viral proteases. Viral proteases are structurally related to two families of cellular proteases, the pepsin-like and trypsin-like proteases. This thesis describes the proteolytic processing of several nonstructural proteins of dengue 2 virus, a representative member of the Flaviviridae, and describes methods for transcribing full-length genomic RNA of dengue 2 virus. Chapter 1 describes the in vitro processing of the nonstructural proteins NS2A, NS2B and NS3. Chapter 2 describes a system that allows identification of residues within the protease that are directly or indirectly involved with substrate recognition. Chapter 3 describes methods to produce genome length dengue 2 RNA from cDNA templates.

The nonstructural protein NS3 is structurally related to viral trypsinlike proteases from the alpha-, picorna-, poty-, and pestiviruses. The hypothesis that the flavivirus nonstructural protein NS3 is a viral proteinase that generates the termini of several nonstructural proteins was tested using an efficient in vitro expression system and antisera specific for the nonstructural proteins NS2B and NS3. A series of cDNA constructs was transcribed using T7 RNA polymerase and the RNA translated in reticulocyte lysates. Proteolytic processing occurred in vitro to generate NS2B and NS3. The amino termini of NS2B and NS3 produced in vitro were found to be the same as the termini of NS2B and NS3 isolated from infected cells. Deletion analysis of cDNA constructs localized the protease domain necessary and sufficient for correct cleavage to the first 184 amino acids of NS3. Kinetic analysis of processing events in vitro and experiments to examine the sensitivity of processing to dilution suggested that an intramolecular cleavage between NS2A and NS2B preceded an intramolecular cleavage between NS2B and NS3. The data from these expression experiments confirm that NS3 is the viral proteinase responsible for cleavage events generating the amino termini of NS2B and NS3 and presumably for cleavages generating the termini of NS4A and NS5 as well.

Biochemical and genetic experiments using viral proteinases have defined the sequence requirements for cleavage site recognition, but have not identified residues within proteinases that interact with substrates. A biochemical assay was developed that could identify residues which were important for substrate recognition. Chimeric proteases between yellow fever and dengue 2 were constructed that allowed mapping of regions involved in substrate recognition, and site directed mutagenesis was used to modulate processing efficiency.

Expression in vitro revealed that the dengue protease domain efficiently processes the yellow fever polyprotein between NS2A and NS2B and between NS2B and NS3, but that the reciprocal construct is inactive. The dengue protease processes yellow fever cleavage sites more efficiently than dengue cleavage sites, suggesting that suboptimal cleavage efficiency may be used to increase levels of processing intermediates in vivo. By mutagenizing the putative substrate binding pocket it was possible to change the substrate specificity of the yellow fever protease; changing a minimum of three amino acids in the yellow fever protease enabled it to recognize dengue cleavage sites. This system allows identification of residues which are directly or indirectly involved with enzyme-substrate interaction, does not require a crystal structure, and can define the substrate preferences of individual members of a viral proteinase family.

Full-length cDNA clones, from which infectious RNA can be transcribed, have been developed for a number of positive strand RNA viruses, including the flavivirus type virus, yellow fever. The technology necessary to transcribe genomic RNA of dengue 2 virus was developed in order to better understand the molecular biology of the dengue subgroup. A 5' structural region clone was engineered to transcribe authentic dengue RNA that contains an additional 1 or 2 residues at the 5' end. A 3' nonstructural region clone was engineered to allow production of run off transcripts, and to allow directional ligation with the 5' structural region clone. In vitro ligation and transcription produces full-length genomic RNA which is noninfectious when transfected into mammalian tissue culture cells. Alternative methods for constructing cDNA clones and recovering live dengue virus are discussed.

Indentification and characterization of a novel CA^(2+)-binding protein in avian erythrocytes

A novel Ca^(2+)-binding protein with Mr of 23 K (designated p23) has been identified in avian erythrocytes and thrombocytes. p23 localizes to the marginal bands (MBs), centrosomes and discrete sites around the nuclear membrane in mature avian erythrocytes. p23 appears to bind Ca^(2+) directly and its interaction with subcellular organelles seems to be modulated by intracellular [Ca^(2+)]. However, its unique protein sequence lacks any known Ca^(2+)-binding motif. Developmental analysis reveals that p23 association to its target structures occurs only at very late stages of bone marrow definitive erythropoeisis. In primitive erythroid cells, p23 distributes diffusely in the cytoplasm and lacks any distinct localization. It is postulated that p23 association to subcellular structures may be induced in part by decreased intracellular [Ca^(2+)]. In vitro and in vivo experiments indicate that p23 does not appear to act as a classical microtubule-associated protein (MAP) but p23 homologues appear to be expressed in MB-containing cells of a variety of species from different vertebrate classes. It has been hypothesized that p23 may play a regulatory role in MB stabilization in a Ca^(2+)-dependent manner.

Binucleated (bnbn) turkey erythrocytes were found to express a truncated p23 variant (designated p21) with identical subcellular localization as p23 except immunostaining reveals the presence of multi-centrosomes in bnbn cells. The p21 sequence has a 62 amino acid deletion at the C-terminus and must therefore have an additional ~40 amino acids at the N-terminus. In addition, p21 seems to have lost the ability to bind Ca^(2+) and its supramolecular interactions are not modulated by intracellular [Ca^(2+)]. These apparent differences between p23 and p21 raised the possibility that the p23/p21 allelism could be the Bn/bn genotype. However, genetic analysis suggested that p23/p21 allelism had no absolute correlation with the Bn/bn genotype.