Cell-free translation of purified virion-associated high-molecular-weight RNA synthesized in vitro by vaccinia virus.

Virion-associated high-molecular-weight (HMW) RNA synthesized in vitro by purified vaccinia virus particles has been translated in a wheat germ cell-free protein synthesizing system. Purified HMW RNA directs the synthesis of translation products which are identical to the translation products made in response to in vitro-synthesized, virion-released 8 to 12S mRNA. The translation of HMW RNA proceeds exclusively through a 5'-terminal cap-mediated initiation step. Furthermore, only one coding sequence is translated per HMW RNA molecule, and that sequence is probably located near the 5' end of the molecule. These conclusions are based on the following results. (i) Sodium dodecyl sulfate--polyacrylamide gel electrophoresis patterns of translation products synthesized in response to HMW RNA and in response to 8 to 12S mRNA were qualitatively identical. (ii) On an equal weight basis, HMW RNA was 25 to 30% as active as 8 to 12S mRNA in stimulating in vitro protein synthesis. (iii) Unmethylated HMW RNA was translated at 10% the efficiency of the methylated form of this RNA. (iv) m7pG inhibited the translation of fully methylated HMW RNA by 90%. (v) After the initiation step of translation was blocked by aurintricarboxylic acid, the rate with which amino acids were incorporated into individual polypeptides decreased in a similar manner for the translation of both HMW RNA and 8 to 12S mRNA. Virion-released 8 to 12S mRNA derived from virion-associated HMW RNA during a chase in the presence of ATP, GTP, and S-adenosylmethionine was also translated. At low RNA concentrations, the derived RNA appeared to stimulate amino acid incorporation more efficiently than the HMW RNA precursor. However, at higher concentrations of this RNA, protein synthesis was severely inhibited.     

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 $\text{in vitro}$.     

Messenger RNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

The virion-associated RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV) synthesizes in vitro two size classes of RNA products similar to those observed in VSV-infected cells. One RNA product sediments at 31S with an approximate molecular weight of 2.1 X 106. The smaller products consist of at least three classes of RNA sedimenting at 17S, 14.5S, and 12S with molecular weights of 0.7 X 106, 0.52 X 106, and 0.37 X 106, respectively. Hybridization experiments show that both the 31S and 12-18S RNA products are complementary to the genome RNA, and that each class is transcribed from different nucleotide sequences. From the molecular weights of the RNA species and the hybridization experiments, it seems that almost the entire VSV genome RNA is transcribed in vitro.     

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.     

Characterization of vesicular stomatitis virus mRNA species synthesized in vitro.

The smallest size class of mRNA (12S) synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus contains two mRNA species of similar molecular weight that code for the viral M and NS proteins. The resolution of these mRNA species was achieved by converting them to duplexes by annealing with the genome RNA, followed by RNase T2 treatment and separation in a polyacrylamide gel. Using this separation technique, the mRNA's were identified by comparing the relative resistance of their syntheses to UV irradiation of the virus. The molecular weights of these two mRNA species calculated as duplex RNAs were smaller than expected. The possible reasons for this discrepancy are discussed.     

Role of intermolecular interactions of vesicular stomatitis virus nucleoprotein in RNA encapsidation

The crystal structure of the vesicular stomatitis virus nucleoprotein (N) in complex with RNA reveals extensive and specific intermolecular interactions among the N molecules in the 10-member oligomer. What roles these interactions play in encapsidating RNA was studied by mutagenesis of the N protein. Three N mutants intended for disruption of the intermolecular interactions were designed and coexpressed with the phosphoprotein (P) in an Escherichia coli system previously described (T. J. Green et al., J. Virol. 74:9515-9524, 2000). Mutants N (Δ1-22), N (Δ347-352), and N (320-324, (Ala)₅) lost RNA encapsidation and oligomerization but still bound with P. Another mutant, N (Ser290→Trp), was able to form a stable ring-like N oligomer and bind with the P protein but was no longer able to encapsidate RNA. The crystal structure of N (Ser290→Trp) at 2.8 Å resolution showed that this mutant can maintain all the same intermolecular interactions as the wild-type N except for a slight unwinding of the N-terminal lobe. These results suggest that the intermolecular contacts among the N molecules are required for encapsidation of the viral RNA.     

Complete nucleotide sequence of the leader rna synthesized in vitro by vesicular stomatitis virus

The complete nucleotide sequence of the leader RNA synthesized in vitro by the Indiana serotype of vesicular stomatitis virus is presented. The sequence was determined by the technique described by Donis-Keller, Maxam and Gilbert (1977) in combination with the standard two-dimensional fingerprint techniques described by Barrell (1971). The leader RNA contains 48 nucleotides variably terminating at the 3′ terminus with cytosine (68%) and adenosine at position 47 (32%). Since the leader RNA is complementary to the 3′ terminal portion of the viral genome RNA, the first 48 nucleotides from the 3′ end of the genome RNA can be deduced. The leader RNA contains repetitive and palindromic sequences with a polypurine sequence at its 3′ terminus. The possible role of some of the sequences is discussed.     

In vitro synthesis of a unique RNA species by a T particle of vesicular stomatitis virus.

A T particle of vesicular stomatitis virus, containing most of the L-gene region, has been isolated. In vitro, these T particles synthesize exclusively a small adenine-rich RNA that is complementary to the T-particle genome. Partial sequence analysis of this small RNA indicates that it is an RNA of unique sequence with a length of approximately 45 nucleotides.     

The 5' terminal structure of the methylated mRNA synthesized in vitro by vesicular stomatitis virus.

The 5' terminal structure of the mRNA synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus in the presence of S-adenosyl-L-methione consists of 7-methyl guanosine linked to 2'-O-methyl adenosine through a 5'-5' pyrophosphate bond as m7G(5')ppp(5')A-m-p ... The alpha and beta phosphated of GTP and alpha phosphate of ATP are incorporated into the blocked 5' terminal structure.     

A unique RNA species involved in initiation of vesicular stomatitis virus RNA transcription in vitro.

Purified virions of vesicular stomatitis virus (VSV) are capable of synthesizing two distinct types of virus-specific RNA in vitro. The first consists of several viral mRNAs which have been previously shown to contain the blocked 5' terminal sequence GpppApApCpApGp and 3' terminal poly(A). The second type of RNA has an unblocked 5' terminus and does not contain poly(A) stretches long enough to bind to oligo (dT)-cellulose columns. It migrates in 20% polyacrylamide gels as a single homogeneous peak with an estimated chain length of 68 nucleotides. Base analysis demonstrated that this small RNA molecule is composed of 48% AMP, 20% CMP, 11% GMP, and 21% UMP. The 5' terminal sequence of the small RNA is ppApCpGp, which appears to be complementary to the 3' terminal sequence of the VSV genome RNA (...PypGpU). These results indicate that this small RNA molecule probably represents the intitiated lead-in RNA segment which is removed during formation of VSV mRNAs by a possible processing mechanism.