Nucleotide sequence complexities, molecular weights, and poly(A) content of the vesicular stomatitis virus mRNA species.

Poly(A)-containing vesicular stomatitis virus mRNA species synthesized in vesicular stomatitis virus-infected cells have been separated into four bands by electrophoresis on formamide-polyacrylamide gels. Two-dimensional fingerprints of ribonuclease T-1 and ribonuclease A digests of the RNA from each band show that they contain unique oligonucleotide sequences as well as 60 to 125 nucleotides of poly(A). The fingerprints were used to determine the nucleotide sequence complexities of RNA from three of the bands. Two contain nucleotide sequences which account completely for their molecular weights (0.70 times 10-6 and 0.55 times 10-6) determined by gel electrophoresis and sedimentation rate, and, therefore, these are radiochemically pure RNA species. The most rapidly migrating band must contain two ro three different RNA species since it has a molecular weight of 0.28 times 10-6, determined by physical methods, and a nucleotide sequence complexity two to three times that expected for a pure RNA species of this size. These data are in complete accord with translational studies (accompanying paper) which show that each of the two pure RNA species codes for a distinct viral protein, whereas the third codes for two viral proteins. From the molecular weight and sequence complexity determinations on mRNA from the bands, we conclude that most of the vesicular stomatitis virus genome is transcribed into discrete mRNA species.     

Nucleotide sequences in the RNA of mammalian leukemia and sarcoma viruses.

The nucleotide sequences in viral RNA from purified murine sarcoma and hamster leukemia viruses (S+H+) from HTG-1 cells and Rauscher leukemia virus (RLV) from JLS-V 9 cells have been examined by polynucleotide agarose affinity chromatography. There is at least one copy of poly(A) sequences per genomic viral RNA molecule. After heat denaturation of genomic viral RNA (S+H+), there are two types of viral subunits for 34S and 28S species: one that contains poly(A) sequences and one that does not. There are no detectable poly(U) tracts in the viral RNA. However, poly(C) sequences and poly(G) tracts were detected in viral RNA, although less poly(G) than poly(C) tracts were observed. In addition, heat-denatured genomic viral RNA has a greater affinity for poly(G) agarose column than native genomic viral RNA.     

Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells.

The cytoplasm of vesicular stomatitis virus (VSV)-infected BHK cells has been separated into a fraction containing the membrane-bound polysomes and the remaining supernatant fraction. Total poly(A)-containing RNA was isolated from each fraction and purified. A 17S class of VSV mRNA was found associated almost exclusively with the membrane-bound polysomes, whereas 14,5S and 12S RNAs were found mostly in the postmembrane cytoplasmic supernatant. Poly(A)-containing VSV RNA synthesized in vitro by purified virus was resolved into the same size classes. The individual RNA fractions isolated from VSV-infected cells or synthesized in vitro were translated in cell-free extracts of wheat germ, and their polypeptide products were compared by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The corresponding in vivo and in vitro RNA fractions qualitatively direct the synthesis of the same viral polypeptides and therefore appear to contain the same mRNA species. By tryptic peptide analysis of their translation products, the in vivo VSV mRNA species have been identified. The 17S RNA, which is compartmentalized on membrane-bound polysomes, codes for a protein of molecular weight 63,000 (P-63) which is most probably a nonglycosylated form of the viral glycoprotein, G. Of the viral RNA species present in the remaining cytoplasmic supernatant, the 14.5S RNA codes almost exclusively for the N protein, whereas the 12S RNA codes predominantly for both the NS and M proteins of the virion.     

Effect of viral infection on host protein synthesis and mRNA association with the cytoplasmic cytoskeletal structure

We studied the association of several eucaryotic viral and cellular mRNAs with cytoskeletal fractions derived from normal and virus-infected cells. We found that all mRNAs appear to associate with the cytoskeletal structure during protein synthesis, irrespective of their 5' and 3' terminal structures: e.g., poliovirus that lacks a 5' cap structure or reovirus and histone mRNAs that lack a 3' poly A tail associated with the cytoskeletal framework to the same extent as capped, polyadenylated actin mRNA. Cellular (actin) and viral (vesicular stomatitis virus and reovirus) mRNAs were released from the cytoskeletal framework and their translation was inhibited when cells were infected with poliovirus. In contrast, actin mRNA was not released from the cytoskeleton during vesicular stomatitis virus infection although actin synthesis was inhibited. In addition, several other conditions under which protein synthesis is inhibited did not result in the release of mRNAs from the cytoskeletal framework. We conclude that the association of mRNA with the cytoskeletal framework is required but is not sufficient for protein synthesis in eucaryotes. Furthermore, the shut-off of host protein synthesis during poliovirus infection and not vesicular stomatitis virus infection occurs by a unique mechanism that leads to the release of host mRNAs from the cytoskeleton.     

Adenylate-Rich Sequences in Vesicular Stomatitis Virus Messenger Ribonucleic Acid

Vesicular stomatitis virus (VSV)-specific messenger ribonucleic acid (mRNA) species contain sequences of adenylate-rich RNA which are more heterogeneous in their migration through sodium dodecyl sulfate-polyacrylamide gels than the corresponding fractions from HeLa cell mRNA. VSV virion RNA contains no adenylaterich sequences. The possible role of such sequences in the mRNA species of a cytoplasmically replicating virus is discussed.     

Translation of Vesicular Stomatitis Messenger RNA by Extracts from Mammalian and Plant Cells

RNA was isolated from polyribosomes of vesicular stomatitis virus (VSV)-infected cells and tested for its ability to direct protein synthesis in extracts of animal and plant cells. In cell-free, non-preincubated extracts of rabbit reticulocytes, the 28S VSV RNA stimulated synthesis of a protein the size of the vesicular stomatitis virus L protein whereas the 13 to 15S RNA directed synthesis of the VSV M, N, NS, and possibly G proteins. In wheat germ extracts, 13 to 15S RNA also directed synthesis of the N, NS, M, and possibly G proteins. Analysis of extracts labeled with formyl [(35)S]methionine showed that the 28S RNA directed the initiation of synthesis of one protein, whereas the 13 to 15S RNA directed initiation of at least four proteins. It is concluded that the 28S RNA encodes only the L protein, whereas the 13 to 15S RNA is a mixture of species, presumably monocistronic, which code for the four other known vesicular stomatitis virus proteins.     

In vitro synthesis of a possible precursor RNA by purified vesicular stomatitis virus.

The RNA products synthesized $\text{in vitro}$ by the virion-associated RNA polymerase of purified vesicular stomatitis virus have previously been shown to contain two distinct 5′-terminal sequences. The mRNA species contain the blocked 5′-terminal G(5′)ppp(5′)A-A-C-A-G sequence and the initiated lead-in RNA segment (approximately 50 bases) contains the unblocked 5′ ppA-C-G sequence. In the present studies, using inosine 5′-triphosphate in place of GTP it is shown that RNA species as large as 14.5S contain an unblocked 5′-ppA-C-(I) sequence indicating that the GTP analogue permits synthesis of a possible precursor of viral mRNA $\text{in vitro}$.     

Gene expression in chemically transformed mouse embryo cells: selective enhancement of the expression of C type RNA tumor virus genes.

Treatment of a nontumorigenic clone of AKR mouse embryo cells in culture with a variety of polycyclic aromatic hydrocarbons has resulted in the development of derivative clones which are highly tumorigenic and exhibit other characteristics of the transformed phenotype. A 3-methylcholanthrene-transformed derivative clone (clone MCA) has been compared to the parent clone (clone 2B) with respect to the abundance and diversity of polysomal poly(A)-containing mRNA sequences. Hybridization kinetic experiments show that the poly(A)-containing sequences of both clones are organized into indistinguishable abundance classes, and that the vast majority of the sequences are common to both the parent and derivative clones. The levels of two specific messenger RNAs (α- and β-globin mRNA) which characterize highly differentiated mouse erythroid cells were much less than 1 molecule per cell in either cell type. Titration of a balanced complementary DNA probe to AKR murine leukemia virus (AKR-MuLV) 70S RNA with purified polysomal poly(A)-containing RNA from both parent and derivative clones shows that approximately 5000 and 1200 viral 35S RNA equivalents are present in the cytoplasm of growing and resting clone MCA cells, respectively. Rapidly growing clone 2B cells contain less than about 30 viral 35S RNA equivalents per cell. Viral specific sequences therefore correspond to members of the high abundance class of poly(A)-containing RNA sequences in clone MCA cells and to the low abundance class of sequences in clone 2B cells. Within the limits of detection, this large increase in abundance is characteristic only of viral specific RNA sequences.     

Mitochondrial poly(A) RNA synthesis during early sea urchin development

The synthesis of mitochondrial messenger RNA during early sea urchin development was examined. Oligo(dT) chromatography and electrophoresis on aqueous or formamide gels of mitochondrial RNA from pulse-labeled embryos showed the presence of eight distinct poly(A)-containing RNA species, ranging in size from 9 to 22 S. Nuclease digestion of these RNAs revealed poly(A) sequences of 4 S size. Using sea urchin anucleate fragments, we were able to demonstrate that all eight messenger RNAs are transcribed from mitochondrial DNA, rather than being transcribed from nuclear DNA and imported into the mitochondria.There was no change in the electrophoretic profile of the eight poly(A) RNAs when embryos were pulsed with [³H]uridine at various times after fertilization. Neither was there any change in the incorporation of [³H]uridine into these species or in the percentage of total newly synthesized mitochondrial RNA that contains poly(A) sequences as development progresses. Even though these RNAs appear to be transcribed at a constant rate throughout early development, they were not detected in mitochondrial polysomes until 18 hr after fertilization.