Relative importance of 7-methylguanosine in ribosome binding and translation of vesicular stomatitis virus mRNA in wheat germ and reticulocyte cell-free systems.

Vesicular stomatitis virus mRNAs with these four types of 5'-termini, (a) m7G5'ppp5'(m)Am, (b) ppp5'(m)Am, (c) m7G5'-ppp5' Am, and (d) G5'ppp5'A, were prepared and their translation and ribosome binding analyzed in wheat germ and reticulocyte cell-free protein synthesis systems. The relative efficiencies of translation of individual vesicular stomatitis virus (VSV) mRNAs having type 2 termini ranged from 23 to 29% of the control (type 1) RNA in the reticulocyte system and 6 to 7% of control RNA in the wheat germ system. A similar difference between the two systems was seen in ribosome-binding experiments in which type 2 RNA formed an 80 S initiation complex with high efficiency (70% of control type 1 RNA) in the reticulocyte system, but with low efficiency (17% of control RNA) in the wheat germ system. Similar differences in the importance of m7G in translation in the two systems were seen when VSV mRNAs synthesized in vitro with type 3 and type 4 termini were analyzed. However, the analysis of type 4 RNA (which was synthesized in vitro in the presence of S-adenosylhomocysteine) was complicated by the presence of abnormally large poly(A) at its 3'-end. Another series of experiments showed that compounds such as 5'pm7G and m7G5'ppp5'Np are potent and specific inhibitors of translation of all types of VSV mRNAs in the wheat germ system (greater than 98% inhibition) but cause less than 20% inhibition of translation in the reticulocyte system. Taken together, all of the results indicate that a 5'-terminal m7G is far more important in translation of VSV mRNAs in the heterologous plant cell-free system than in the reticulocyte lysate system.     

Inhibition of protein synthesis in vesicular stomatitis virus infected Chinese hamster ovary cells: role of virus mRNA-ribonucleoprotein particle

Although host protein synthesis is preferentially inhibited, there is a steady decline in the ability of Chinese hamster ovary (CHO) cells infected with vesicular stomatitis virus (VSV) to synthesize both host and viral proteins. We previously reported finding an mRNA-ribonucleoprotein particle (mRNP) that contained all five VSV mRNAs and viral N protein exclusively. This particle apparently regulates translation by sequestering a majority of the VSV mRNA made late in infection and thus rendering it unavailable for protein synthesis. In the present investigation the mRNP was also shown to inhibit in vitro protein synthesis in rabbit reticulocyte and wheat germ lysates programmed with mRNA isolated from VSV-infected cells. The synthesis of eIF-2 X GTP X Met-tRNA (ternary) complex, the first step in initiation of protein synthesis, was markedly inhibited by the mRNP. The inhibition was partially reversed by addition of purified eIF-2 to the inhibited lysate or ternary complex formation reaction. These results indicate a dual role of the mRNP in regulating protein synthesis during infection. Nucleocapsid also inhibited in vitro protein synthesis, although this inhibition was not reversed by eIF-2. Nucleocapsid did not inhibit ternary complex formation in vitro. Consequently, nucleocapsid may also regulate in vivo protein synthesis, but by a mechanism different from the mRNP.     

Shut-off of actin biosynthesis in adenovirus serotype-2-infected cells

Adenovirus produces a dramatic shut-off of host protein synthesis after infection of HeLa cells. The level of actin messenger RNAs remained relatively unchanged after viral infection, when assayed by in vitro translation and two-dimensional gel electrophoresis analysis of the proteins or hybridization of the total cytoplasmic RNAs to the human actin gene. The distribution of actin mRNA in the polyribosomes is altered after adenovirus infection, with small polyribosomes and monoribosomes of the infected cells occupied by actin messages untranslatable in a rabbit reticulocyte lysate. The large polyribosomes still retain enough functional mRNAs to provide significant levels of actin protein in a rabbit reticulocyte in vitro translation system. In contrast, in homologous infected cell lysates, the translation of exogenous actin mRNA is greatly reduced when compared to uninfected HeLa cell lysates. In nuclease-treated uninfected or infected HeLa cell-free extracts, translation of viral mRNA is equally efficient and higher than that of actin mRNA. Thus, translational regulatory mechanisms which include inactivation of a part of the actin mRNA population accompanied by displacement to small polysomes and/or virus-induced modification of the cellular translational machinery to discriminate against cellular actin mRNA seem to account for the sharp reduction in actin protein synthesis of adenovirus-infected cells.     

N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.     

Characterization and translation of methylated and unmethylated vesicular stomatitis virus mRNA synthesized in vitro by ribonucleoprotein particles from vesicular stomatitis virus-infected L cells.

Ribonucleoprotein particles isolated from extracts of vesicular stomatitis virus (VSV) -infected L cells synthesized in vitro four classes of polyadenylated RNA sedimenting at 29S, 19S, 17S, and 13S. When synthesized in vitro in the presence of the methyl donor S-adenosyl methionine, these RNA species contained the following 5'-terminal structures: (i) m7G5ppp5'AmpAp(70%) ; (ii) m7G5'ppp5'AmpAmpNp (20%) and (iii) pppAp (10%). In the presence of the methylation inhibitor S-adenosylhomocysteine, however, the mRNA contained the 5'-terminal structures G5'ppp5'Ap (80%) and pppAp (20%). The mRNA's synthesized in vitro were translated in the homologous ascites and the heterologous wheat embryo cell-free systems. In both, the products were shown by sodium dodecyl sulfate gel electrophoresis and by immunoprecipitation to contain all five viral proteins, L, G, N, NS, and M. The presumed precursor to the G protein (G*) was also identified by fingerprint analysis. Methylated VSV mRNA was more active in protein synthesis than unmethylated mRNA in both the ascites system and the wheat embryo systems. Addition of S-adenosylmethionine stimulated translation of unmethylated mRNA in the wheat embryo but not in the ascites extract. S-adenosylhomocysteine, however, by preventing mRNA methylation inhibited the translation of unmethylated VSV mRNA in both systems. The mRNA methylating activity present in wheat embryo S-30 extracts was recovered in the ribosome-free supernatant fraction (S-150) and was insensitive to the protein synthesis inhibitor pactamycin.     

Differential post-translational modification of human type I keratins synthesized in a rabbit reticulocyte cell-free system

The translation products synthesized in a rabbit reticulocyte lysate system, from total cellular mRNA from the human epithelial cell-line ME-180, have been examined. Keratin proteins are prominent among these translation products, and they precisely coelectrophorese in sodium dodecyl sulfate-polyacrylamide gels with keratins purified from the cells. Type-I, acidic, keratins which are acetylated in vivo, are also acetylated by the reticulocyte lysate. Examination by two-dimensional electrophoresis, of two acidic keratins known to be phosphorylated in vivo reveals that only one of these proteins is phosphorylated in the lysate system. Phosphorylation of this protein occurs after release of the completed polypeptide chain from the ribosome. The protein phosphorylated by the lysate is known to be the only ME-180 phosphokeratin modulated by cyclic AMP, reflecting in vitro the differential modification of ME-180 keratins in vivo.     

In Vitro Synthesis of Proteins by Membrane-Bound Polyribosomes from Vesicular Stomatitis Virus-Infected HeLa Cells

Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P(88). In vitro synthesized protein P(88) does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes.     

Multiple levels of regulation of protein synthesis at fertilization in sea urchin eggs

Fertilization of sea urchin eggs results in a large stimulation of protein synthesis. This increase in protein synthesis is mediated by the mobilization of stored maternal mRNA (mRNPs) into polysomes, but the details of the molecular mechanisms which regulate this process are not well understood. Using a sea urchin egg cell-free translation system, evidence has been obtained which indicates that the capacity to initiate protein synthesis on new mRNAs is limited. Addition of exogenous mRNAs failed to stimulate overall protein synthesis, whereas supplementing the system with a nuclease-treated reticulocyte lysate, an S-100 supernatant fraction, or purified eIF-2 stimulated nearly twofold. In addition, the levels of 43 S preinitiation complexes containing a 40 S ribosomal subunit and methionyl-tRNA were increased at pH 7.4 compared to pH 6.9, or when reticulocyte S-100 was added. However, other experiments showed clearly that mRNA availability may also regulate translation in the sea urchin egg. Sea urchin lysates only stimulated poorly the nuclease-treated reticulocyte lysate system, and the mRNPs in the sea urchin lysate did not bind to reticulocyte 43 S preinitiation complexes. Since purified sea urchin egg mRNA was active in both assays, the bulk of sea urchin mRNA must be masked in the egg, and remain masked in the in vitro assays. Thus, protein synthesis appears to be regulated at both the level of mRNA availability and the activity of components of the translational machinery.     

NH2-terminal acetylation of Dictyostelium discoideum actin in a cell-free protein-synthesizing system

We present here a new method for inhibiting protein acetylation in a rabbit reticulocyte cell-free protein-synthesizing system. This procedure utilizes S-acetonyl coenzyme A, a nonreactive acetyl-CoA analogue, as an inhibitor of the NH2-terminal protein acetyltransferase in this lysate. With this procedure, we can make, in vitro, Dictyostelium discoideum actin which is 85% nonacetylated but fully translated. With the fully translated but nonacetylated actin as a substrate, the actin can be almost completely acetylated post-translationally in an acetyl-CoA-dependent system after the actin has left the ribosome. Using formylated and nonformylated [35S]Met-tRNAfMet as a source of label and in conjunction with detailed peptide mapping experiments with trypsin and thermolysin, the in vitro acetylation is shown to occur at the NH2 terminus of the newly synthesized actin. Furthermore, the initiator methionine residue, contrary to expectation, is not cleaved off but remains stable for at lest 50 min. thus, in the acetylating reticulocyte lysate system, the primary complete translation product in actin synthesis is Ac-Met-Asp and not Ac-Asp.     

Heterogeneity of vesicular stomatitis virus particles: implications for virion assembly

Vesicular stomatitis virus (VSV) particles formed at early times after infection contain only one-third the amount of viral glycoportein (G protein), relative to the major internal structural proteins M and N, as is found in particles released later. These "early" particles also have a lower density in equilibrium sucrose gradients than do those formed later; however, the sedimentation velocity and specific infectivity of these two classes of particles are the same. VSV-infected cells also release virus-like particles which sediment considerably faster than authentic virions and contain a higher-than-normal proportion of the VSV G protein relative to internal VSV proteins. These particles have a reduced specific infectivity but a normal density in sucrose gradients. All classes of VSV virions contain a constant proportion of M and N polypeptides. The ratio of G protein to M or N protein, in contrast, can vary over a sixfold range; this implies that an interaction between a precise number of surface G proteins with either of the underlying M and N proteins is not a prerequisite for budding of infectious viral particles from the cell surface.     

Vesicular stomatitis virus mRNA and inhibition of translation of cellular mRNA--is there a P function in vesicular stomatitis virus?

Infection of animal cells by vesicular stomatitis virus (VSV) results in inhibition of translation of cellular mRNA. We showed previously that, in BHK cells infected by the Glasgow isolate of VSV Indiana, this is due to competition during the initiation step of protein synthesis of viral and cellular mRNA for a constant, limiting number of ribosomes. We show here that infection of the same cells with the San Juan isolate of VSV resulted in a more rapid shutoff of host protein synthesis and that this was paralleled by a more rapid accumulation of viral mRNA. Extending our conclusion that shutoff is due to mRNA competition, we show further that the average size of polysomes translating viral and cellular mRNA was threefold smaller in cells infected by VSV San Juan than by VSV Glasgow, which, in turn, was about one-half that of uninfected cells. In all cases, cellular and viral mRNA's which encoded the same-sized polypeptides were found on the same-sized polysomes, a result indicating that the efficiency of translation of both types of mRNA's is about the same in the infected cell. Also, there was no preferential sequestration of viral or cellular mRNA's in ribonucleoprotein particles. Additional correlations between the levels of viral mRNA's and the inhibition of protein synthesis came from studies of three other wild-type VSV strains and also from studies with Vero and L cells. In particular, the rate of shutoff of L-cell protein synthesis after infection by any VSV isolate was slower than that in BHK cells, and this was correlated with a slower rate of accumulation of viral mRNA. VSV temperature-sensitive mutants which synthesized, at the nonper-missive temperature, no VSV mRNA failed to inhibit synthesis of cellular proteins. Stanners and co-workers (C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11:273-281, 1977) claimed that VSV mutant R1 inhibited synthesis of L cell protein synthesis less rapidly than did its parent wild-type strain HR. They concluded that this effect was due to a mutation in an unspecified VSV protein, “P.” We found, in both L and BHK cells, that R1 infection resulted in a slightly slower inhibition of cellular mRNA translation than did HR infection and that this was correlated with a slightly reduced accumulation of VSV mRNA. The level of VSV mRNA, rather than any specific VSV protein, appeared to be the key factor in determining the rate of shutoff of host protein synthesis.