ASSOCIATION OF THE NONSTRUCTURAL PROTEIN NS(S) OF UUKUNIEMI VIRUS WITH THE 40S RIBOSOMAL-SUBUNIT

The small RNA segment (S segment) of Uukuniemi (UUK) virus encodes two proteins, the nucleocapsid protein (N) and a nonstructural protein (NS(s)), by an ambisense strategy. The function of NS(s) has not been elucidated for any of the bunyaviruses expressing this protein. We have now expressed the N and NS(s) proteins in Sf9 insect cells by using the baculovirus expression system. High yields of both proteins were obtained. A monospecific antibody was raised against gel-purified NS(s) and used to study the synthesis and localization of the protein in UUK virus-infected BHK21 cells. While the N protein was detected as early as 4 h postinfection (p.i.), NS(s) was identified only after 8 h p.i. Both proteins were still synthesized at high levels at 24 h p.i. The half-life of NS(s) was about 1.5 h, while that of the N protein was several hours. Sucrose gradient fractionation of [S-35]methionine-labeled detergent-solubilized extracts of infected BHK21 cells indicated that NS(s) was firmly associated with the 40S ribosomal subunit. This association took place shortly after translation and was partially resistant to 1 M NaCl. NS(s) expressed by using the T7 vaccinia virus expression system, as well as in vitro-translated NS(s), was also associated with the 40S subunit. In contrast, in vitro-translated N protein was found on top of the gradient. Immunolocalization of NS(s), in UUK virus-infected cells, by using an affinity-purified antibody showed a granular cytoplasmic staining. A very similar pattern was seen for cells expressing NS(s) from a cDNA copy by using a vaccinia virus expression system. No staining was observed in the nuclei in either case. Furthermore, NS(s) was found neither in virions nor in nucleocapsids isolated from infected cells. In vivo labeling with P-32i indicated that NS(s) is not phosphorylated. The possible function of NS(s) is discussed in light of these results.