Mouse hepatitis virus persists in cultures of a subline (designated LM-K) of mouse LM cells but produces a lytic infection in L-2 cells. Persistence in the LM-K cells was not accompanied by production of ts mutants or of soluble anti-MHV factors. Infectious center assay demonstrated an approximately 500-fold lower level of infectibility by MHV of the LM-K cells as compared to L-2 cells. On an infected cell basis, production levels of infectious progeny and viral RNA were comparable between the two cell lines. The extent of virus-induced cell-cell fusion, however, was markedly reduced in the LM-K cells. Cell-mixing experiments showed that both infected L-2 and LM-K cells have the capacity of fusing with neighboring uninfected L-2 cells but not with uninfected LM-K cells. This suggests that the decreased level of fusion observed in the LM-K infection is due not to absence of viral fusion protein at the cell surface, but rather to an inherent resistance of the LM-K cell membrane to MHV-induced fusion. It is believed that such fusion resistance in LM-K cells moderates virus dissemination throughout the culture, thereby contributing to a state of virus persistence.

The expression vectors pINIII-A and pINIII (Ipp p5) were used to construct plasmids which direct the synthesis in Escherichia coli of the Kirsten ras viral (v-Ki-ras) and human cellular (c-Ki-ras) oncogene products as fusion proteins containing 9 and 10 extra amino acids, respectively, at their N termini. Authenticity of the bacterially produced proteins was determined by immunoprecipitation and immunoblot analyses with ras-specific monoclonal antibodies. After induction with isopropyl-I8-D-thiogalactopyranoside, the viral protein represented approximately 20% of the total cellular protein. The majority of the protein was found in the postsonication low-speed centrifugation pellet. The synthesized viral protein was active in GTP binding, as judged by autophosphorylation and photoaffinity labeling assays. * Corresponding author. t Present address: Creative Biomolecules, Hopkinton, MA 01748. expression vector pINIII-A2 was digested with EcoRI, and the ends were made flush by treatment with DNA polymerase I (large fragment), and the vector was then digested with HindIII. The ras-containing fragment was ligated to the digested pINIII-A2, and the ligated DNA was used to 302 on July 6, 2020 by guest http://jvi.asm.org/ Downloaded from EXPRESSION OF KIRSTEN ras PROTEINS 303 transform JA221 cells. The A2 reading frame of the expres-