Morphogenesis of reovirus as defined by the Fields panel of temperature sensitive mutants

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Hazelton, Paul Russell
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The use of amber mutations of bacteriophage have allowed complicated pathways of assembly to be dissected for bacteriophage systems. However, viral assembly continues to be poorly understood with the eucaryotic viruses. Because of the value of amber mutations with bacteriophage the current studies into the assembly pathways of reovirus were initiated using the Fie ds panel of temperature sensitive ts mutants of reovirus. One problem with investigations involving the Fields panel was the inability to clearly identify the ts phenotype in some clones. Increasing restrictive temperature by 0.5 to 1.0 $\sp\circ$C effected additional reductions in the titre of infectious progeny obtained at restrictive temperatures, reducing the ratio of infectious progeny between permissive and restrictive cultures by up to three orders of magnitude. Using this data it was possible to clearly identify the ts phenotype in a number of clones which were refractive to earlier study. The prototype clones for recombination groups E and I were evaluated. In addition to clearly associating the ts lesion to the appropriate gene segment it was possible to confirm that neither clone was able to establish a productive infection at restrictive temperatures. Neither tsE320, nor tsI138 produced dsRNA at restrictive temperature. Further, the clone tsI138 was identified as having a slow growth phenotype, consistently producing reduced levels of dsRNA and titres of infectious progeny at permissive temperatures than the wild type parent and other mutant clones. Ultrastructural investigations with this clone indicated that the viral inclusions at permissive temperatures were rare and disorganized. Ts mutations were identified on two separate gene segments with the clone tsA279. The M2 gene segment, which encodes the major outer capsid protein $\mu$1, was associated with a blockade in transmembrane transport of restrictively assembled virions. Four point mutations were identified in the genetic sequence for this segment, each effecting a change in the primary structure of the encoded protein. Two mutations, in proximity with each other, changed prolines to serenes at amino acid residues 306 and 315, and disrupted predicted secondary structure in amphipathic regions by changing proposed $\alpha$- helices to $\beta$-sheets. The other two mutations were also in proximity with each other, effecting primary structure changes from proline to threonine and glutamate to lysine at residues 673 and 687, respectively. These mutations affected predicted positive charge in $\phi$, the carboxy terminus proteolytic fragment of $\mu$1, and may affect the ability of the fragment to interact with host membranes during transmembrane transport. The second lesion, in the L2 gene segment, was associated with the production of aprimary core particle. This core particle was defective for both minor proteins $\mu$2 and $\lambda$2. This is the first report of a primary core particle comprised of the core proteins $\lambda 1$, $\lambda 3$, and $\sigma 2$. The existence of this primary core particle has profound implications concerning the assembly processes of reovirus.