Translation extracts were prepared from various strains of Saccharomyces cerevisiae. The translation of mRNA molecules in these extracts was cooperatively enhanced by the presence of 5'-terminal cap structures and 3'-terminal poly(A) sequences. These cooperative effects could not be observed in other translation systems such as those prepared from rabbit reticulocytes, wheat germ, and human HeLa cells. Because the yeast translation system mimicked the effects of the cap structure and poly(A) tail on translational efficiency seen in vivo, this system was used to study cap-dependent and cap-independent translation of viral and cellular mRNA molecules. Both the 5' noncoding regions of hepatitis C virus and those of coxsackievirus Bi conferred cap-independent translation to a reporter coding region during translation in the yeast extracts; thus, the yeast translational apparatus is capable of initiating cap-independent translation. Although the translation of most yeast mRNAs was cap dependent, the unusually long 5' noncoding regions of mRNAs encoding cellular transcription factors TFIID and HAP4 were shown to mediate cap-independent translation in these extracts. Furthermore, both TFIID and HAP4 5' noncoding regions mediated translation of a second cistron when placed into the intercistronic spacer region of a dicistronic mRNA, indicating that these leader sequences can initiate translation by an internal ribosome binding mechanism in this in vitro translation system. This finding raises the possibility that an internal translation initiation mechanism exists in yeast cells for regulated translation of endogenous mRNAs.

The unique oncogene carried by the McDonough strain of feline sarcoma virus (SM-FeSV), called v-fms, directs the synthesis of a set of related glycoproteins, called gP 180gag-fms, gp 140fms, and gp 120fms. We have prepared antibodies to these proteins and used indirect immunofluorescence techniques on viable SM-FeSV transformed cells to demonstrate that fms-specific determinants are expressed on the external surface. The fms-specific fluorescence co-localized with clathrin and was detectable in clathrin-coated pits and endocytotic vesicles. Two cell surface labeling methods indicated that gp140fms was the only fms-related protein on the cell surface. In view of the relationship between the erbB oncogene product and the epidermal growth factor receptor, and the fact that growth factor receptors utilize clathrin-coated pits in endocytosis, we believe the gp140fms transforming protein of SM-FeSV also could function as an analog of a growth factor receptor.

Genetic and biochemical analyses of the 5' noncoding region of poliovirus have indicated the importance of this region in both translation and amplification of the viral RNA. The role of the cellular machiner required for these events is just beginning to be revealed. Using an RNA gel retention assay, we have identified a cellular 50-kDa protein that forms a specific complex with a stable stemloop structure present in the viral 5' noncoding region. The formation of the RNA,-protein complex is dependent on the availability of free sulfhydryl groups in the protein. The possible involvement of this RNA-protein complex in the regulation of viral gene expression is discussed.Polioyirus, a member of the Picornaviridae family, is a plus-stranded RNA virus that multiplies in the cytoplasm of the infected human or monkey host cell. The 7500-nucleotide (nt) RNA molecule encodes a 220-kDa polyprotein that is cleaved by virally encoded proteases to yield the processed structural and nonstructural proteins (1). The 5' and 3' noncoding regions of the viral RNA contain sequences that mediate efficient translation and positive-strand and negative-strand amplification. Although certain sequence elements involved in these events have been identified by site-directed mutagenesis of an infectious cDNA clone (reviewed in ref.2), the identification of cellular and viral proteins that bind to these sequences has only recently been actively pursued. For example, a sequence element in the 747-nt 5' noncoding region from nt 320-631 has been found to bind cellular ribosomes internally, explaining the unusual cap-independent translation of the viral RNA (3). Subsequently, cellular proteins have been identified that bind to this region and are being examined for their role in mediating internal ribosome binding (4, 5).We Konarska and Sharp (9). Briefly, 32P-labeled RNA molecules (1 pmol; 108 cpm/ pug), made in vitro by T7 RNA polymerase, were incubated in 10 ,uI (total volume) ofbinding buffer into which extracts from uninfected or polioyirus-infected HeLa cells had been added. Seventy micrograms of total protein was present in each binding reaction mixture unless otherwise specified. Poly-(A,C,U) (Sigma)