Characterization of two recombination-complementation groups of Uukuniemi virus temperature-sensitive mutants.
With the aim of isolating temperature-sensitive (ts) mutants defective in virus maturation or glycoprotein transport, Uukuniemi virus, a bunyavirus, was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine. Out of 13 initial clones unable to grow at 39 degrees C (non-permissive temperature), five mutants which grew to titres above 10(7) p.f.u./ml at 32 degrees C (permissive temperature) were selected for further studies. The mutants fell into two coinciding recombination-complementation groups. Three group I mutants ( ts7 , 8 and 12) and two group II mutants ( ts6 and 11) synthesized all three RNA segments and were able to form the corresponding nucleoproteins at 39 degrees C. Thus, members of these two recombination groups had a RNA-positive phenotype. All five mutants showed immunofluorescence when cells were stained at 39 degrees C using a double-staining technique employing monoclonal antibodies against the glycoproteins G1 or G2, and polyclonal antibodies against the nucleoprotein, N. We have previously shown that in cells infected with wild-type virus both the G1/G2 and the N proteins accumulate in the Golgi complex, the site of virus maturation. In cells infected with ts12 , accumulation of G1 and G2, but not N protein, was observed in the Golgi complex at 39 degrees C. The N protein was found evenly scattered in the cytoplasm, suggesting lack of interaction between the G1/G2 and N proteins. With ts6 and 11, G1 and G2 appeared to accumulate and aggregate in the endoplasmic reticulum (ER) at 39 degrees C. The location of the N protein coincided with that of the aggregated glycoproteins, suggesting that the N protein interacted with G1/G2 already in the ER. Thus, these mutants may prove valuable tools in studying the mechanism of Uukuniemi virus maturation.
D002043:Bunyaviridae / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D005814:Genes, Viral / Q000187:drug effects; D005816:Genetic Complementation Test; D006023:Glycoproteins / Q000096:biosynthesis; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D009154:Mutation; D009698:Nucleoproteins / Q000096:biosynthesis; D010641:Phenotype; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic / Q000187:drug effects; D013696:Temperature; D014764:Viral Proteins / Q000096:biosynthesis
D006023:Glycoproteins; D009698:Nucleoproteins; D012367:RNA, Viral; D014764:Viral Proteins; D008769:Methylnitronitrosoguanidine