In Vitro Translation of Uukuniemi Virus-Specific RNAs: Identification of a Nonstructural Protein and a Precursor to the Membrane Glycoproteins

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Abstract
Virus-specific RNA species from Uukuniemi virus-infected chick embryo cells were isolated and fractionated by sucrose gradient centrifugation. In addition to 3 RNA species cosedimenting with the 3 viral RNA segments L (29S), M (23S) and S (17S), a 4th major RNA species, sedimenting at about 12S (S2), was found early in the infection. Annealing experiments indicated that the cytoplasmic L and M RNA species consisted of plus and minus strands, with the plus strands in slight excess. Most S1 RNA was of negative polarity; S2 was of positive polarity. The S2 RNA specifically annealed to the virion S RNA segment, indicating that it is transcribed from this segment. In vitro translation of the individual RNA species in micrococcal nuclease-treated cell-free reticulocyte extracts [rabbit] showed that an mRNA cosedimenting with the virion M RNA directed the synthesis of a virus-specific 110,000 dalton polypeptide (p110). This polypeptide could be immunoprecipitated with antiserum prepared against purified virions. When translation was carried out in the presence of dog pancreas microsomes, p110 was absent. Instead, an immunoprecipitable polypeptide band, with a MW of about 70,000 and migrating between the virion surface glycoproteins G1 and G2, was observed. Thus the glycoproteins are probably synthesized as a precursor (p110), which during translation is cleaved roughly in the middle to yield G1 and G2. The 12S RNA species directed the synthesis of the nucleocapsid protein and a novel polypeptide with an apparent MW of about 30,000. The latter was not precipitated with antivirion serum and was absent from lysates programmed with the corresponding RNA fraction from a mock-infected extract. Since, in addition, it was not found in purified virions and was present in the cytoplasm of infected cells but not in uninfected cells, it probably represents a nonstructural polypeptide.