Mammalian reoviruses have been used as a model to study the control of host cell protein synthesis following infection. The reovirus genome is composed of 10 segments of double stranded RNA each of which, with one exception, encodes a single protein. The three serotypes of the virus show marked differences in their effects on host cell protein synthesis following viral infection. Serotypes 2 and 3 give pronounced inhibition and serotype 1 has little effect. Genetic studies using intertypic reassortants have mapped this property to the S4 gene of the virus which encodes the major outer capsid polypeptide σ3. The S4 gene of serotypes 1 and 3 have been sequenced and show 96 % homology at the amino acid level. Full length cDNA clones of the S4 gene of serotypes 1 and 3 (S4-1 and S4-3) were used to develop in vitro and in vivo assay systems to study the mechanism underlying the differential effects on host cell protein synthesis of the corresponding σ3 proteins. The ability of σ3 to inhibit host cell protein synthesis was investigated in two in vitro translation systems: the rabbit reticulocyte lysate system and S-10 cell extracts prepared from uninfected and type 1 and type 3 infected L-cells. Σ3 had no effect on either the translation of B-actin when used as a marker for eukaryotic protein synthesis, or endogenous protein synthesis in either of these systems. In vivo assay systems made use of the HIVLTR/tat inducible reporter system to drive σ3 expression. The effects of σ3 on the expression of two reporter genes, chloramphenicol acetyltransferase (CAT) and B-galactosidase were investigated in transient in vivo assay systems. Σ3 expressed from S4-3 cDNA caused a significant stimulation of reporter gene expression. In contrast σ3 from S4-1 cDNA had no effect on gene expression, suggesting a domain of S4-3 was responsible for this stimulation. In an attempt to domain map this property two hybrids were constructed, one containing the S' 800 base pairs of S4-1 and the 3' 400 base pairs of S4-3 (HY-1), and the other, HY-3, the converse of this. Σ3 synthesised from HY-1 stimulated reporter gene expression whereas that from HY-3 had no effect. Σ3 was readily detected in cells transiently expressing either the parental type 3 S4 cDNA or the HY-1 hybrid by immunoprecipitation and immunofluorescence. By contrast only very small amounts of oi were detected when either the S4 cDNA from type 1 or the HY-3 were used. Pulse-chase experiments indicated that the σ3 from type 1 virus is less stable than the type 3 protein and that the domain responsible for this stability is in the 3' terminus of the S4 gene. This domain is also that shown by others to be responsible for the ability of σ3 to bind dsRNA.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:293814 |
| Date | January 1991 |
| Creators | Martin, Patricia E. M. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/108094/ |
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