Molecular genetics of hantaviruses: Cloning, sequencing, expression, and morphogenesis.
|Title:||Molecular genetics of hantaviruses: Cloning, sequencing, expression, and morphogenesis.|
|Abstract:||Molecular characterization of the Seoul 80-39 virus, a prototype of the Seoul serotype, was carried out by cloning and sequencing. The large (L) genomic RNA segment is 6530 nucleotides long. The viral complementary-sense RNA contains a single open reading frame (ORF) from the AUG codon at nucleotide positions 37-39 to the UAA stop codon at nucleotide positions 6490-6492. This ORF could encode a polypeptide of 2151 amino acids (246,662 D) which likely corresponds to the L protein detected in purified viral particles (Elliot et al., 1984) and is assumed to be an RNA-dependent RNA polymerase molecule (Schmaljohn and Dalrymple, 1983). The M RNA segment is 3651 nucleotides long. A single ORF was detected in the viral complementary-sense RNA. This ORF could encode a polypeptide of 1133 amino acids which is cotranslationally processed into viral glycoproteins G1 and G2. The S genomic RNA segment was characterized from the 3$\sp\prime$ end to nucleotide 1332. One long ORF was detected from the AUG codon at nucleotide positions 43-45 to the UAA stop codon at nucleotide positions 1330-1332. This ORF has a capacity to encode a 48 kD protein which corresponds to the viral nucleocapsid (N) protein. A comparison of coding properties of the tripartite genomes of various hantaviruses allowed construction of the evolutionary tree for the Hantavirus genus. The second part of the study was focused on the maturation of Hantaan 76-118 virus glycoproteins G1 and G2 virus morphogenesis. Glycoproteins G1 and G2 form a complex. Experiments with the glycosylation inhibitor brefeldin A indicate that complex formation between the two glycoproteins occurs in the ER. Resistance of both G1 and G2 to endoglycosidase D suggests a structure with more than five mannose residues. Processing of a high mannose structures ((Man)$\sb8$(GlcNAc)$\sb2$ to (Man)$\sb7$(GlcNAc)$\sb2$ or (Man)$\sb6$(GlcNAc)$\sb2$) may take place in the cis-Golgi by the enzyme $\alpha$-mannosidase I. Virus budding was observed only in Hantaan virus-infected cells, in the trans-Golgi network. When the Golgi apparatus was destroyed by brefeldin A treatment, assembly of virus particles was not observed. The third project was to express nucleocapsid proteins of Seoul 80-39 virus and Hantaan 76-118 virus using baculovirus recombinants. Analysis of recombinant virus-infected Sf9 cell lysates by SDS-PAGE showed that N proteins were expressed in high levels and were identical to virion associated N proteins in size and antigenicity. (Abstract shortened by UMI.)|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|