Alternatives for the initiation of translation.

New evidence of exceptions to the scanning mechanism for the initiation of translation has been recently obtained. These data suggest that ribosomes can bind and initiate internally on certain mRNAs without having to scan from the 5' end.

The beta 1 sodium channel subunit modifies the interactions of neurotoxins and local anesthetics with the rat brain IIA alpha sodium channel in isolated membranes but not in intact cells.

Mammalian brain sodium channels consist of an alpha subunit and two smaller beta subunits. The role of the beta 1 subunit in modulating ligand interactions at these channels was examined using a cell line stably expressing human beta1 and rat brain IIA alpha subunits. Coexpression of the beta 1 subunit had no effect on the potencies of sodium channel blockers in inhibiting whole cell [3H]batrachotoxinin A benzoate ([3H]BTX) binding or veratridine-stimulated [14C]guanidinium influx. Coexpression of the beta 1 subunit also had no effect on the potencies of alpha scorpion toxin, brevetoxin, or RU 39568 in stimulating [14C]guanidinium influx. By contrast, coexpression of the beta 1 subunit had dramatic effects on ligand interactions in isolated membranes. In isolated membranes of cells expressing only the alpha subunit, the neurotoxins had no stimulatory effect on [3H]BTX binding and the potencies of local anesthetic-like channel inhibitors were 10-100-fold lower than those at native sodium channels. Whereas in membranes of cells coexpressing the beta 1 subunit, the neurotoxins increased [3H]BTX binding 30-fold and the potencies of the sodium channel inhibitors closely matched those found at native sodium channels. These findings indicate that the beta 1 subunit is not required for the binding of sodium channel activators or inhibitors but rather, that the beta 1 subunit may stabilize the alpha subunit in a functional conformation thereby allowing detection of these interactions in disrupted membranes.

The herpes simplex virus type 1 ICP6 gene is regulated by a 'leaky' early promoter.

Expression from the promoter for the large subunit (ICP6) of the ribonucleotide reductase encoded by herpes simplex virus type 1 (HSV-1) has been examined. Using the lacZ reporter gene fused in-frame with ICP6 regulatory sequences to assay expression quantitatively, we showed that the ICP6 promoter responded very weakly to the alpha-transinducing factor (TIF) in the absence of all other viral gene products, but much more strongly to immediate early proteins. Similar patterns of regulation were observed when the reporter gene construct was located at two different positions within the the viral genome or in a stably transfected Vero cell line. Infection of the stably transfected cells with various HSV-1 mutants identified ICP0 as the major transactivator of the ICP6 promoter.

Nonpermissive infection of lymphoblastoid cells by vesicular stomatitis virus. I. Synthesis and function of the viral transcripts.

The human B-lymphoblastoid cell line Raji is nonpermissive for infection by vesicular stomatitis virus (VSV) (Nowakowski et al. (1973) J. Virol. 12, 1272-1278). Viral-specific transcription begins immediately after infection, but Raji cells synthesize only about one-twentieth as much viral RNA as is synthesized by a permissive host. The viral primary transcripts appear to be unstable in Raji cells when prevented from engaging in protein synthesis by the addition of cycloheximide. The messages are undermethylated in the 5'-terminal cap structure and have a relatively short 3'-polyadenylate tail. Nevertheless, the subcellular distribution of the messages indicates that many of these RNAs are present in large polyribosomes. Analyses of the effects of a temperature-sensitive mutation in the viral matrix protein indicate that mRNA synthesis in Raji cells is limited only by the amount of available nucleocapsid templates and not by a specific defect in transcription.

Nonpermissive infection of lymphoblastoid cells by vesicular stomatitis virus. II. Effect on viral morphogenesis.

The human B-lymphoblastoid cell line Raji is nonpermissive for infection by vesicular stomatitis virus (VSV). The VSV particles released from Raji cells display a more heterogeneous distribution in equilibrium sucrose density gradients than particles released from BHK cells. The particles released from Raji cells contain approximately one-half to one-third as much viral matrix protein, relative to the nucleocapsid protein, as is normal. They also contain a higher proportion of the unglycosylated form of the G protein. The particles released from Raji cells are unstable and many disintegrate in the growth medium. Most of them deform when subjected to ultracentrifugation prior to fixation. The ratio of plaque-forming units to physical particles is much lower for the virions released from Raji cells.

Deletion of the herpes simplex virus type 1 ribonucleotide reductase gene alters virulence and latency in vivo.

The role of herpes simplex virus type 1 (HSV-1)-encoded ribonucleotide reductase (RR) has been investigated in mice and guinea pigs using a mutant from which 90% of the large subunit of the enzyme was deleted. The RR mutant was extremely impaired in its ability to induce external vaginal lesions or to cause death in mice following intracerebral, intraperitoneal, or intravaginal inoculation, or in guinea pigs following intraperitoneal or intravaginal inoculation. The RR mutant replicated poorly in the vagina of mice and guinea pigs when compared with the parental virus. Neither infectious nor latent virus was recovered from the trigeminal ganglia of mice or from the dorsal root ganglia of mice and guinea pigs after inoculation with the RR mutant. Using the polymerase chain reaction, RR mutant DNA was, nevertheless, detected in the dorsal root ganglia of guinea pigs. These studies suggest that HSV-1 RR is essential for virulence and may also play a role in the recovery of reactivatable latent virus from ganglia in both mice and guinea pigs.

Internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA yields a second protein.

In vitro translation of a mixture of the vesicular stomatitis virus (VSV) polyadenylated mRNAs yielded a previously undetected protein with a molecular weight of approximately 7,000 (7K protein). Hybrid-arrested translation demonstrated that both the 7K protein and the VSV phosphoprotein (P protein) were encoded by the P protein message. Immunoprecipitation of the 7K protein with monoclonal antiserum directed against the P protein indicated that the two products were encoded in the same open reading frame. A protein of approximately the same size was immunoprecipitated from cytoplasmic extracts of VSV-infected cells by both the polyclonal and monoclonal antisera, and it is likely that it was a previously unrecognized viral gene product. Translational mapping of the P protein mRNA in vitro indicated that the 7K protein was encoded in the 3' one-third of the sequence. The synthesis of the 7K protein in vitro was unaffected by hybrid arrest conditions which blocked the 5' two-thirds of the mRNA and inhibited synthesis of the P protein. These results imply that the ribosomes bind and initiate translation internally on the P protein mRNA at a site located hundreds of nucleotides downstream from the capped 5' end.

Characterization of the internal initiation of translation on the vesicular stomatitis virus phosphoprotein mRNA.

Internal initiation of translation on the vesicular stomatitis virus (VSV) phosphoprotein (P) mRNA leads to the synthesis of a second protein [Herman, R. C. (1986) J. Virol. 58, 797-804]. Characterization of this phenomenon shows that initiation at the 5'-proximal and internal AUG codons has different optima for mono- and divalent cations in the reticulocyte lysate. Whereas 5' initiation is stimulated by increasing concentration of K+ over the endogenous level, internal initiation is inhibited. Internal initiation is much less sensitive to the effects of the cap analogue 7mGpppG in both the reticulocyte lysate and the wheat-germ extract under conditions that reduce 5'-proximal initiation to only about 4-5% of the control level. These results imply that 5'-proximal and internal initiations are distinct biochemical processes.

Synthesis of respiratory syncytial virus RNA in cell-free extracts.

A cell-free system has been used to synthesize respiratory syncytial virus (RSV) RNA in vitro. Polyadenylated species representing all size classes of RSV mRNAs were labelled. Some of the labelled RNA was seen in a CsCl gradient at the density characteristic of negative-strand viral nucleocapsids. Experiments using a thiotriphos-phorylated nucleotide indicate that RNA chains are initiated de novo in the cell-free system.

Conditional synthesis of an aberrant glycoprotein mRNA by the internal deletion mutant of vesicular stomatitis virus.

The internal deletion mutant (DI-LT) derived from the heat-resistant strain of vesicular stomatitis virus synthesizes an aberrant polyadenylated mRNA (G*) containing a transcript of the partially deleted polymerase gene covalently linked to the 3' end of the glycoprotein message (R. C. Herman and R. A. Lazzarini, J. Virol. 40:78-86, 1981). The heat-resistant polymerase appears to play a role in the synthesis of the abnormal G* RNA. The synthesis of G* correlated directly with the presence of the heat-resistant L protein on the defective interfering particle template. Chimeric defective interfering particles produced by passaging DI-LT with a helper virus that encodes the wild-type vesicular stomatitis virus polymerase did not synthesize G*. The subsequent passage of the chimeric DI-LT with a heat-resistant helper virus restored the ability to synthesize the G* transcript. These results imply that the regulatory signals normally present at the vesicular stomatitis virus G/L intercistronic boundary may be preserved in DI-LT. These sequences are only conditionally functional because they are recognized correctly by the wild-type but not by the heat-resistant polymerase.

Analysis of the message-sequence content of the pulse-labeled poly(A)+ heterogeneous nuclear RNA from HeLa cells by cDNA-excess hybridizations.

The message-sequence content of pulse-labeled poly(A)+ HeLa heterogenous nuclear RNA (hnRNA) has been examined by hybridizations to an excess of message cDNA. Control experiments show that the message cDNA accurately reflects the sequence distribution of the complex mixture of poly(A)+ messages present in the HeLa cytoplasm. Pulse-labeled poly(A)+ molecules in both the lamina-associated and shnRNA fractions contain message sequences, and approximately 65% of the poly(A)-adjacent hnRNA sequences are homologous to the 3' ends of mRNA. The majority of the pulse-labeled hnRNA molecules contain abundant message sequences. By use of these techniques it is also shown that some pulse-labeled polyadenylated message sequences are still synthesized in the presence of the adenosine analogue 5,6-dichloro-beta-D-ribofuranosylbenzimidazole under conditions where little or no new cytoplasmic mRNA is produced.

Nucleotide sequence of an aberrant glycoprotein mRNA synthesized by the internal deletion mutant of vesicular stomatitis virus.

The transcriptionally active internal deletion mutant (DI-LT) of vesicular stomatitis virus synthesizes an abnormal mRNA (G*) containing a transcript of the remnant polymerase gene covalently linked to the 3' end of the glycoprotein message (R.C. Herman and R.A. Lazzarini, J. Virol. 40:78-86, 1981). A complementary DNA copy of the 3' end of the G* transcript was molecularly cloned and then chemically sequenced. The results showed that the deletion removed the last 54 nucleotides of the normal glycoprotein gene, the intergenic dinucleotide, and all but the last 258 nucleotides of the polymerase gene. The sequence of DI-LT at the deletion site was compared to that of the transcriptionally inactive DI-LT2 particle.

In vivo repair of the single-strand interruptions contained in bacteriophage T5 DNA.

Bacteriophage T5 is known to contain several unique single-strand interruptions in only one strand of the duplex DNA. Analysis of labeled parental phage DNA from infected Escherichia coli shows that these nicks are repaired in vivo to yield intact double-stranded molecules. Sealing begins at about 6 min after infection and is independent of DNA replication. Repair may be an ordered process that starts at a unique end of the molecule.

Aberrant glycoprotein mRNA synthesized by the internal deletion mutant of vesicular stomatitis virus.

The internal deletion mutant (DI-LT) derived from the heat-resistant strain of vesicular stomatitis virus synthesized an aberrant polyadenylated mRNA in vivo and in vitro. No normal glycoprotein message could be detected among the in vivo transcription products. The abnormal RNA contained a transcript of the partially deleted polymerase gene covalently linked to the 3' end of the glycoprotein message. The polyadenylate is located at the 3' end of the molecule and is most probably encoded by the remnant polymerase gene polyadenylation signal. This aberrant RNA may be synthesized because of either a failure to terminate transcription at the end of the glycoprotein gene or an inability to process an abnormal polycistronic precursor.

Vesicular stomatitis virus RNA polymerase can read through the boundary between the leader and N genes in vitro.

Triphosphorylated in vitro transcripts of vesicular stomatitis virus were selected by mercury-Sepharose chromatography using adenosine-5'-O-(3-thiotriphosphate) as an affinity probe. Numerous RNAs ranging from less than 47 up to several hundred nucleotides in length were detected. Some of these contain the leader RNA covalently linked to transcripts of the N gene. A comparison of the published genomic sequences at the estimated termination sites of several of these RNAs reveals some homology with the sequence present at both the end of the leader and polymerase genes.

In vitro characterization of a herpes simplex virus type 1 ICP22 deletion mutant.

We report the construction of a deletion mutant (del22Z) that is unable to synthesize any detectable messenger RNA or protein products from the herpes simplex virus type 1 (HSV-1) immediate early ICP22 gene upon infection. The del22Z deletion mutant lacks all but 18 nucleotides of the ICP22 coding sequence and carries the bacterial lacZ gene at the site of the deletion. No other known open reading frames or flanking sequences were disrupted. Del22Z was able to infect Vero cells productively but was severely restricted in human and rodent cells that were permissive for the parental HSV-1(F). The yield of del22Z was not enhanced significantly, either by increasing the multiplicity of infection or by increasing the duration of the infection. There was a prolonged expression of some early gene products and a delayed appearance of some late gene products in both permissive and restrictive cells. This phenotype of cell-line restricted growth and alteration of the normal gene expression cascade maps specifically to the ICP22 coding region.

Increased synthesis of polycistronic mRNA associated with increased polyadenylation by vesicular stomatitis virus.

Electron microscopy suggested that the mRNA produced in vitro by tsG16(I), a temperature-sensitive mutant of vesicular stomatitis virus, contained an increased proportion of polycistronic mRNAs. Using hybrid selection, we found that the poly(A)+ mRNA synthesized in vitro by tsG16(I) contained approximately two to three times more polycistronic mRNA than did poly(A)+ mRNA synthesized in vitro by the parental wild-type (wt) virus. The increase in polycistronic mRNA occurred at all intergenic junctions examined. In vitro, tsG16(I) has an increased polyadenylation phenotype and a temperature-sensitive transcriptase activity that appear to be due to different mutations. Partial revertants of tsG16(I), which have lost the aberrant polyadenylation phenotype but retain the in vitro thermosensitive transcriptase, produced wt amounts of polycistronic mRNA. This suggested that the increased production of polycistronic mRNA by tsG16(I) may be associated with the increased polyadenylation phenotype of this mutant. These data further support the hypothesis that an increase in size of poly(A) tracts is associated with increased production of polycistronic mRNA.

Message and non-message sequences adjacent to poly(A) in steady state heterogeneous nuclear RNA of HeLa cells.

Highly purified steady state heterogeneous nuclear RNA from HeLa cells has been prepared by a new procedure. Detergent-washed nuclei are disrupted in 0.4 M ammonium sulfate, which also disociated contamination polysomes. The hnRNA remains bound to chromatin, which can be pelleted by gentle centrifugation. Ribonuclease inhibitors permit the preparation of very high molecular weight nuclear RNA. The hnRNA was cleaved with alkali. The poly(A)-containing fragments were separated from those containing oligo(A), and a cDNA copy was prepared. Hybridization of this nuclear cDNA to cytoplasmic mRNA showed that the scarce (complex) message sequences make up a larger proportion of nuclear RNA than of cytoplasmic RNA. In addition, at least 30% of the poly(A)-adjacent sequences in nuclear RNA have no apparent counterparts in the cytoplasm. cDNA prepared from hnRNA sedimenting faster than 45S under denaturing conditions gives similar results, showing the presence of both message and non-message sequences in very large transcripts. cDNA complementary to mRNA was separated into the abundant and scarce sequences, and hybridized separately to the poly(A)-adjacent sequences in nuclear RNA. The hybridization of the abundant sequence cDNA was used to set an upper limit on possible cytoplasmic contamination. Hybridization of the scarce cytoplasmic sequences are represented in nuclear RNA approximately once per cell.

Defective interfering particle generated by internal deletion of the vesicular stomatitis virus genome.

The genome structure of the long, truncated defective interfering particle derived from the heat-resistant strain of vesicular stomatitis virus has been examined. Stocks of this defective interfering particle are shown to contain several different species having information primarily from the 3' half of the vesicular stomatitis virus genome; the proportions of these components vary depending on the passage history of the stock. The two most abundant types have been identified and characterized. One has complementary 5' and 3' termini and consequently appears as a circular molecule when examined by electron microscopy. The other cannot circularize and remains linear. The circular forms are consistently 8 to 10% longer than the linear molecules. Rapid sequencing analyses reveal that both forms retain the 5' parental viral terminal sequence, but only the linear form retains the parental 3'-terminal sequence which is the complement of the 5' end. Hybridization experiments and electron microscopic analyses indicate that the linear form has retained 320 to 350 nucleotides of the 5' parental sequence and was probably generated by an internal deletion of the vesicular stomatitis virus genome.

Polycistronic vesicular stomatitis virus RNA transcripts.

A procedure to enrich for the sequences present at the junction between the linked messages in the polycistronic RNAs symthesized in vitro by vesicular stomatitis virus (VSV) is described. Analyses of these sequences show that they contain a precise transcript of both the intercistronic dinucleotide and the pentanucleotide 5'--C-U-G-U-U--3', common to the 5'-end of all VSV cistrons, covalently linked to the 3'-side of the intervening poly(A). The data strongly suggest that the VSV transcriptase polyadenylylates the mRNAs and can then resume direct and precise transcription of the genome-without reinitiation and without skipping nucleotides.