Viral Messenger RNA Research Articles

High-resolution preparative gel electrophoresis: Separation and recovery of functional messenger RNA species

We report a simple method for the simultaneous separation and recovery of large quantities of discrete viral messenger RNA (mRNA) species from a total cytoplasmic extract. The method utilizes an electronic control device that directs continuous cycles of simultaneous electrophoresis and elution of RNA followed by automated collection and fractionation. This method of separation gives high recovery (80–90%) of intact RNA species. The RNAs isolated by this method are shown to be discrete as measured by analysis in analytical gels and to be functional as measured by their ability to direct synthesis of proteins in vitro. Analysis of products of the in vitro translation of the isolated RNA species indicates that it is possible to resolve RNAs with very similar mobilities by this method.

In Vitro translation of rabies virus messenger RNAs

RNA isolated from rabies virus-infected cells directed the in vitro synthesis of the five major rabies virus structural proteins.

Polyamines stimulate suppression of amber termination codons in vitro by normal tRNAs.

Polyamines, such as spermine and spermidine, are able to stimulate reading of amber termination codons on viral messenger RNAs in vitro. This phenomenon is not due to an overall increase of error frequency during translation, but to a specific effect on a normal tRNA that is present in various eukaryotic cell preparations. The enhancement of reading of termination codons by normal tRNAs should be of major importance for the expression of specific genes in eukaryotic cells.

RNA primers and the role of host nuclear RNA polymerase II in influenza viral RNA transcription.

Influenza viral RNA transcription in the infected cell is inhibited by alpha-amanitin, a specific inhibitor of the host nuclear RNA polymerase II. Because viral RNA transcription in vitro catalysed by the virion-associated transcriptase is greatly enhanced by the addition of a primer dinucleotide, ApG or GpG, we have proposed that viral RNA transcription in vivo requires initiation by primer RNAs synthesized by RNA polymerase II. In addition, because we did not detect any capping and methylating enzymes in virions, we have proposed that the 5' terminal methylated cap found on in-vivo viral messenger RNA (mRNA) is derived from the putative primer RNAs. Our recent experiments have proved these two hypotheses. Purified globin mRNAs were shown to stimulate viral RNA transcription in vitro very effectively. The resulting transcripts directed the synthesis of all the non-glycosylated virus-specific proteins in cell-free systems. Other eukaryotic mRNAs were also active as primers. The presence of a 5' terminal methylated cap structure in the priming mRNA was required for its priming activity. Thus, with globin mRNA, removal of the cap eliminated essentially all of its priming activity, and much of this activity could be restored by enzymically recapping the globin mRNA. Using globin mRNA containing 32P only in its cap, we demonstrated that the 5' cap of the globin mRNA primer was physically transferred to the viral RNA transcripts during transcription. Gel electrophoretic analysis suggested that, in addition to the cap, about 10-15 other nucleotides were also transferred from the globin mRNA to the viral RNA transcripts. A mechanism for the priming of influenza viral RNA transcription by globin mRNA is proposed. Initial experiments strongly suggest that priming by capped host mRNAs also occurs during the synthesis of viral mRNA in vivo.

Protein synthesis in vaccinia virus-infected cells in the presence of amino acid analogs: A translational control mechanism

A rapid and efficient block of protein synthesis occurs in EAT or L cells exposed to the amino acid analogs ( p-fluorophenylalanine and canavanine) and further infected by vaccinia virus. Cells preinfected for 30 min and then exposed to the analogs do not show this inhibition of protein synthesis which appears to result from a factor associated with the input virions. It is shown that this translational block occurs also on viral early messenger RNAs since they appear to be normally transcribed in the presence of the analogs, as judged by the incorporation of labeled uridine into total cytoplasmic RNA, the size of the poly(A) plus fraction and its capacity to be translated in vitro. The absence of inhibition of viral messenger RNA translation in the preinfected cells further exposed to the amino acid analogs is likely to result from the rapid synthesis of some early protein either involved in a cascade mechanism of translation or releasing the effect of the inhibitory factor associated with the input vaccinia virions. Separation by two-dimensional gel electrophoresis revealed that most of the viral proteins analyzed were not synthesized according a cascade mechanism of translation. The synthesis of one to two viral proteins may occur through the latter mechanism or alternatively these proteins may be modified after their translation by some virus coded enzyme.

On the origin of the polyhedral protein of Autographa californica nuclear polyhedrosis virus Isolation, characterization, and translation of viral messenger RNA

Virus-specific messenger RNA was isolated 24 hr postinfection from Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV) by means of hybridization with viral DNA that was coupled to cellulose. Viral mRNA was analyzed on isokinetic sucrose gradients and on polyacrylamide gels containing 98% formamide. Viral mRNA was also translated in an in vitro protein-synthesizing system derived from wheat germ. One product comigrated with purified AcNPV polyhedral protein (MW 30,000) and precipitated with antibodies against AcNPV polyhedral protein. These results favor the hypothesis that the polyhedral protein is a virus-coded protein.

Unusual features in the nucleotide sequence of a cDNA clone derived from the common region of avian sarcoma virus messenger RNA.

We have constructed a recombinant plasmid containing a 700-base pair (bp) cDNA copy of the common region present at the 3' end of Schmidt-Ruppin avian sarcoma virus (ASV) 21S mRNA. The cDNA was inserted into plasmid pBR322 at the Pst I site by the G-C tailing method. A restriction map of the cloned insert from a recombinant plasmid pSRI indicates that it corresponds to the 3' end of the ASV genome. R-loop analysis with ASV genomic RNA indicates that the insert is colinear with the ASV genome over most of its length. The sequence of 331 bp at the 3' end of the DNA insert was determined and shows that the insert contains extra sequences not found at the 3' end of ASV genomic RNA. Following the terminally redundant sequence of 20 bp that has been found at the extreme 3' end of genomic RNA is a sequence of 79 bp that is almost identical to that located immediately next to the 20-bp repeat at the 5' end of ASV genomic RNA. This is followed by 18 bp of unique sequence, possibly of host origin. The structure of the clone suggests that ASV mRNA may differ from genomic RNA at its 3' end and that 21S mRNA is transcribed from integrated ASV DNA and contains at its 3' end sequences derived both from the 5' end of the ASV genome and from host DNA adjacent to the site of integration. The presence of termination codons in all three reading frames suggests that the common region probably does not contain coding sequences. However, the presence of sequences that resemble probable promoter sites supports the possibility that this region may be involved in the regulation of transcription.

Isolation of foot-and-mouth disease virus messenger RNA from membrane-bound polyribosomes and characterization of its 5′ and 3′ termini

A membrane-bound fraction prepared from foot-and-mouth disease virus (FMDV)-infected baby hamster kidney cells included [ 3H]uridine-labeled 37 S viral messenger RNA (mRNA) and a 20 S RNA species that is partly ribonuclease resistant. The mRNA appeared to contain both a polyadenylic acid (poly (A)) and a polycytidylic acid (poly (C)) tract as demonstrated by its binding to oligo(dT)- and oligo(dG)-cellulose, respectively. Paper chromatography of an RNase T 2 digest of the [ 3H]adenosine-labeled poly(A) fragment from mRNA showed that the poly(A) is at the 3′ end of the mRNA and is approximately 45 nucleotides long. Digestion of 32P-labeled FMDV mRNA with RNase A and T 2 yielded pUp as the 5′ terminus; RNases A and T 2 digested FMD virion RNA lacked this structure.

Inhibitory effect of methylated derivatives of guanylic acid for protein synthesis with reference to the functional structure of the 5′-‘cap’ in viral messenger RNA

Guanylic acid modified variously with methyl groups on base or sugar moieties were synthesized chemically and their inhibitory effects on protein synthesis were tested in a wheat germ cell-free system using mRNAs from cytoplasmic polyhedrosis virus and tobacco mosaic virus. The confronting dinucleotide m 7G 5′ pppA that corresponds to the most simple ‘cap’ structure of an eukaryotic mRNA is a strong inhibitor of protein synthesis, but non-methylated G 5′ pppA or G 5′ ppA is not inhibitory. The strong inhibitory effect is observed only by 7-methylguanylic acid (pm 7G). Among 11 derivatives of pG, the most effective inhibitors are methylated at the 7-position. Further methylation at the other position sometimes cancels the inhibitory effect. Although pm 7G carries a positively charged base, other nucleotides which carry a plus charged base (1-methyladenylic acid and 2-methylthio-7-methylinosinic acid) were not inhibitory. Thus, methylation at the 7-position on guanylic acid is specifically required for the inhibitory effect. Addition of pm 7G was inhibitory for the formation of the initiation complex for eukaryotic protein synthesis. These results suggest that the ‘cap’ component containing 7-methylguanylic acid in viral mRNA participates during protein synthesis, especially in its initial steps. Protein synthesis in a bacterial cell-free system was not inhibited by addition of m 7GpppA or pm 7G when either TMV RNA or phage MS2 RNA was used as an mRNA.

Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs

The studies described here demonstrate that the expression of many early adenovirus mRNAs is dependent upon the activity of a pre-early viral product. This viral gene product is defective in adenovirus 5 host range (Ad hr) group I mutants. Adenovirus 5 host range mutants were previously isolated by their ability to replicate in the adenovirus 5-transformed human embryonic cell line 293 and by their inability to replicate efficiently in HeLa cells (Harrison, Graham and Williams, 1977). The group I complementation class of host range mutants has been mapped by marker rescue between 0 and 4.4 units (Frost and Williams, 1978). We have used the S1 nuclease gel technique to examine the expression of early mRNA after infection of HeLa cells with Ad5 hr group I and II mutants. The Ad5 hr group II mutants stimulate the synthesis of a wild-type pattern of early mRNAs. In contrast, infection of HeLa cells with Ad5 hr group I mutants gives rise to only two early mRNAs. These mRNAs map from 1.5–4.4 units, or in the same region as the Ad5 hr group I mutations. Since infection of HeLa cells with Ad5 hr group I mutants was defective for synthesis of cytoplasmic mRNAs complementary to three early regions in the right half of the genome and to the early region 4.5–11.0 units, we also analyzed nuclear RNA from these cells by the S1 nuclease gel technique for the presence of precursor RNA chains. Nuclear precursors were not detected in Ad5 hr group I-infected HeLa cells, suggesting that the gene product defective in these mutants is required for synthesis of stable nuclear RNA from the three early regions in the right half of the genome and from the early region 4.5–11.0 units.

The effect of ultraviolet radiation on the primary transcription of influenza virus messenger RNAs

The primary transcription of individual mRNA species coded in vivo by influenza virus was studied after varying doses of ultraviolet radiation. The decline in synthesis of each messenger RNA showed single-hit kinetics and target sizes for the inhibition of each were derived. These values approximated the molecular weights of the individual genome segments indicating that each segment possessed its own promoter site for the transcription of its corresponding mRNA.

About 30% of minute virus of mice RNA is spliced out following polyadenylation

MANY viral and eukaryotic messenger RNAs have been shown to contain blocks of sequence which, although adjacent to one another in the RNA, are copied from non-contiguous regions of the genome that are joined together post-transcriptionally. This phenomenon of RNA splicing has been shown to occur during the processing of several DNA and RNA tumour viruses1–7 and cellular8–10 mRNAs. The mechanism by which splicing is carried out has not been elucidated, and any direct approach is complicated by the elaborate splicing patterns found in the systems which have been examined so far. We have extended these studies of RNA splicing to a potentially simpler viral system, the minute virus of mice (MVM), a non-defective member of the parvovirus group. The members of this group are considered to be among the simplest of the animal viruses, and comprise an icosahedral virion made from three structural protein species, containing one linear single-stranded DNA chromosome of ∼1.5 × 106 molecular weight (for review see ref. 11). The MVM DNA molecule contains a stable hairpin duplex at its 5′-end and a 3′-terminal structure suitable for priming complementary strand synthesis in vitro12,13. Little is known about the transcription process in parvo viruses. The only cytoplasmic mRNA species found for adeno-associated virus (AAV), a member of the defective subgroup, is a transcript of 70% of the genome which haS been shown by in vitro translation to code for the three capsid proteins14. The one study of transcription carried out in the non-defective subgroup has identified an analogous RNA species in Kilham rat virus (KRV)-infected cell cytoplasm which is complementary to 70% of the viral strand and sediments as a single component at about 18S (ref. 15). We report here our attempts to determine the sequence arrangement of MVM RNA by examining, in the electron microscope, hybrids formed between the abundant species of nuclear and cytoplasmic poly(A)+ RNA and single-stranded genomic DNA. We have found that about 30% of MVM RNA is spliced out following polyadenylation.

Segments of simian virus 40 DNA spanning most of the leader sequence of the major late viral messenger RNA are dispensable.

A highly specific procedure for the isolation of deletion mutants is described. The size and location of the deletions can be predetermined. By this method a series of deletion mutants mapping within and near the untranslated 5' leader sequence of the late 16S mRNA of simian virus 40 have been isolated. The boundaries of the deletions have been accurately determined by DNA sequence analysis. The deletions range from 20 to 223 nucleotides. All these deletions mutants are viable and grow without helper virus. The largest of these deletions removes the entire leader sequence except for six nucleotides at the 3' end that are probably involved in covalent linkage with the 5' end of the body of the mRNA located 937 nucleotides away on the genome. Three of the deletion mutants remove the 5' end of the leader that normally bears the cap structure of the mRNA. A large segment immediately preceding the leader sequence is also removed in one of these mutants, ruling out the generation of the 5' end of the mRNA via initiation of transcription at this point. The circularization of linear infecting DNA producing the DNA of the deletion mutants proceeds mainly by way of blunt end ligation in vivo.

Uncapping of viral messenger RNA by phosphodiesterase of fibroblast plasma membranes

Isolated plasma membranes from mouse fibroblast lines 3T3 and its tranformant SV-3T3 contain a phosphodiesterase (oligonucleotidase, E.C. 3.1.4.19; nucleotide pyrophosphatase, E.C. 3.6.1.9) that splits capped and methylated messenger RNA obtained from both reovirus and vesicular stomatitis virus. The isolated membranes are free of demonstrable ribonuclease activity and split the mRNA to produce 7-methyl guanosine diphosphate as a product. With ATP as substrate for the phosphodiesterase enzyme, the product is AMP. Synthetic caps, AMP, ADP and ATP, but not cyclic AMP, can compete with the substrate p-nitrophenyl thymidilic acid. A possible regulatory role on messenger translation is proposed.

Decay of vesicular stomatitis virus mRNAs in Vivo

Abstract The stability of vesicular stomatitis virus (VSV) messenger RNA during infection of Chinese hamster ovary (CHO) cells was investigated using the temperature-sensitive mutant, tsG114. By incubating infected cells at the nonpermissive temperature (39°), RNA synthesis was blocked and VSV mRNA decayed rapidly, as determined by sucrose density gradient analysis. Each of the five VSV mRNAs was also found to decay functionally at approximately the same rate suggesting that the differential rates of accumulation of VSV mRNAs observed in vivo are due to differential rates of transcription of specific VSV genes.

Enhanced expression of viral polypeptides and messenger RNA in dimethyl sulfoxide and bromodeoxyuridine-treated Friend erythroleukemic cells

The intracellular virus-specific macromolecular changes induced by dimethyl sulfoxide (Me 2SO) and/or bromodeoxyuridine (BUdR) have been analysed in the Friend erythroleukemic cell clone 745 A 19 (FLC). These cells, which were transformed in vivo by the Friend virus, are arrested in an undifferentiated state but can be induced to differentiate in vitro by Me 2SO. We have shown that treatment of FLC with 2% Me 2SO for 4 days enhances the extracellular virus production and, concomitantly, increases by about 4-fold the intracellular virus-specific polyribosomal RNA. The simultaneous addition to FLC of Me 2SO and BUdR (the latter is known to inhibit the Me 2SO-induced differentiation) brings about an even greater increase in both viral polypeptides and viral messenger RNA (mRNA). As a consequence of treatment with both drugs, new viral nucleotide sequences are expressed, as revealed by nucleic acid hybridization studies. Moreover, it appears that two p30-like virus-specific intracellular polypeptides, instead of one single p30, are expressed in FLC treated with Me 2SO and BUdR. These data also support the idea that gene sequences different from those present in the original Friend virus are expressed as a consequence of the combined treatment. It is concluded that the main site of action of both drugs in this cell system, is at the level of transcription of viral RNA and it is suggested that the drugs may act upon cell differentiation by modulating differently the expression of integrated viral genomes.

Nuclease activation by double-stranded RNA and by 2′,5′-oligoadenylate in extracts of interferon-treated chick cells

Cell-free extracts were prepared from primary chick embryo cells that had been pretreated overnight with chick interferon or with mock preparations of interferon. After the addition of double-stranded RNA (dsRNA) to these extracts, the synthesis of vesicular stomatitis virus (VSV) messenger RNA and its corresponding proteins was measured under conditions of coupled transcription and translation directed by purified virus. The addition of dsRNA to extracts of mock interferon-treated cells did not affect VSV-directed transcription or translation. However, in extracts of interferon-treated cells, the rate of RNA accumulation was inhibited by about 50% and that of the viral proteins by about 95% relative to the rates in control extracts. Furthermore, no intact VSV mRNA was detectable among the transcription products made in extracts of interferon-treated cells under these conditions. These results contrasted with those obtained in the absence of dsRNA, where the interferon-mediated inhibition of protein synthesis was less extreme, and where both sets of transcription products were intact and fully functional. The differences could be ascribed, at least in part, to a dsRNA-mediated increase in the rate of mRNA degradation that occurred only in extracts of interferon-treated cells. Enhancement of mRNA breakdown was also observed when protein synthesis was inhibited by the addition of pppA 2′p 5′A 2′p 5′A and higher oligomers (2′,5′-oligoadenylate) to extracts of either interferon-treated or mock interferon-treated cells. (These novel oligonucleotides are synthesized in interferon-treated cell extracts by an enzyme that requires dsRNA for activation.) Under a variety of conditions, enhanced mRNA degradation correlated with the inhibition of protein synthesis mediated by 2′,5′-oligoadenylate but not by other inhibitors. Thus, the data suggest that the inhibition of protein synthesis by dsRNA in extracts of interferon-treated chick cells is due, at least in part, to mRNA breakdown catalyzed by a nuclease that is activated by 2′,5′-oligoadenylate.

Vesicular stomatitis virus mRNA methylation in Vivo: Effect of cycloleucine, an inhibitor of S-adenosylmethionine biosynthesis, on viral transcription and translation

Cycloleucine is a potent and specific inihibitor of S-adenosylmethionine biosynthesis and subsequent RNA methylations in Chinese hamster ovary (CHO) cells. When added in the course of an infection of CHO cells with vesicular stomatitis virus (VSV), cycloleucine does not significantly modify the rate of viral RNA and protein synthesis or the production of mature particles. Under the same conditions cycloleucine will reduce by 83% the methyolation of newly synthesized polyadenylated 11–16 S viral RNA. The mode of action of cycloleucine and cycloheximide are clearly distinguishable under these conditions. In contrast when cells are treated with cycloleucine, immediately after virus adsorption, the effect on viral RNA synthesis is very similar to that of cycloheximide. That is, a 90% inhibition in the production of viral messenger RNAs is observed along with a complete disappearance of newly synthesized 38 S RNA species, suggesting that secondary transcription is inhibited. Viral protein synthesis and virus production are similarly inhibited during this period. The undermethylated mRNAs of VSV synthesized in the presence of cycloleucine are 80% undertranslated as compared to mRNAs synthesized in untreated cells. Our results suggest that mRNA methylation in VSV is required for translation in vivo. The mode of action of cycloleucine during the infectious process is discussed.

A comparative study of three closely related cytoplasmic polyhedrosis viruses

A cytoplasmic polyhedrosis virus (CPV) from the pine caterpillar, Dendrolimus spectabilis, was compared with Japanese isolates of closely related viruses from the silkworm, Bombyx mori, and gypsy moth, Lymantria dispar. The sizes of the viral RNA genome segments were almost identical, although the CPVs from D. spectabilis and L. dispar could be distinguished from the silkworm virus by a small size difference (0.03 × 10 6 daltons) in one segment. The same viruses were also distinguishable by RNA homology differences of 25–50% measured by the reannealing of 3H-labeled single-stranded viral messenger RNA (synthesized in vitro) to heat-denatured viral double-stranded RNA. Antigenic differences were also detected by gel immunodiffusion tests. CPVs of D. spectabilis and L. dispar were indistinguishable by these criteria.

Intervening polyadenylate sequences in RNA transcripts of vesicular stomatitis virus

Purified and partially resolved vesicular stomatitis virus (VSV) messenger RNA has been annealed to the VSV genomic RNA and visualized in the electron microscope under conditions in which duplex regions have a wider image width than single-stranded RNA. The locations of the intercistronic boundaries between the messages have been mapped on the VSV genome. The contour of the double-stranded regions is occasionally interrupted by looped-out single-stranded RNA. The loops are comprised of post-transcriptionally synthesized polyadenylate. Most of these structures are found at the intercistronic boundaries and covalently bridge adjacent message sequences. In this paper, we discuss the possible significance of these loops.