With the aim of isolating temperature-sensitive () mutants defective in virus maturation or glycoprotein transport, Uukuniemi virus, a bunyavirus, was mutagenized with -methyl-′-nitro--nitrosoguanidine. Out of 13 initial clones unable to grow at 39 °C (non-permissive temperature), five mutants which grew to titres above 10 p.f.u./ml at 32 °C (permissive temperature) were selected for further studies. The mutants fell into two coinciding recombination-complementation groups. Three group I mutants (7, 8 and 12) and two group II mutants (6 and 11) synthesized all three RNA segments and were able to form the corresponding nucleoproteins at 39 °C. Thus, members of these two recombination groups had a RNA-positive phenotype. All five mutants showed immunofluorescence when cells were stained at 39 °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 12, accumulation of G1 and G2, but not N protein, was observed in the Golgi complex at 39 °C. The N protein was found evenly scattered in the cytoplasm, suggesting lack of interaction between the G1/G2 and N proteins. With 6 and 11, G1 and G2 appeared to accumulate and aggregate in the endoplasmic reticulum (ER) at 39 °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.


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