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Biomedical subjects

R A Lazzarini

Publications and source records attributed to R A Lazzarini.

At least 91 records · Page 5Linked to original sources
PubMed1982

In vitro transcription of vesicular stomatitis virus: initiation with GTP at a specific site within the N cistron.

In vitro transcripts of vesicular stomatitis virus (VSV) were either 5'-terminally labeled by incorporation of [beta-(32)P]GTP or were selected on Hg-agarose after incorporation of gamma-thio-GTP. Capped RNAs ranged in size from 23 nucleotides, the shortest capped RNA detected, to full-length message size. The 5'-terminal sequences corresponded to those of N message and to a small amount of NS message. Approximately 14% of the capped N gene transcripts were terminated at positions 86 to 90 of the VSV genome, giving rise to specific, 36 to 40-nucleotide-long, capped RNA species. The GTP-initiated RNAs were short with a predominant 28-nucleotide-long RNA species. A minor portion was as large as mRNAs. Nucleotide sequence analyses of the short RNA revealed that it was specifically initiated at positon 91 of the VSV genome, 41 nucleotides within the N cistron. This corresponds exactly to the site where transcription of the 40-nucleotide-long, capped RNA terminated. Initiation with GTP at position 91 occurred at approximately the same frequency as termination of the capped RNA at position 90, suggesting that intracistronic initiation at position 91 may depend upon termination of transcription of the 5'-proximal region and therefore may be sequential. This unique RNA represents the first transcript of VSV which was initiated at an intracistronic site with GTP, and may also represent the first example of a transcript derived from a stop/start mechanism of VSV transcription in vitro. Although initiation occurred frequently at the beginning of the N cistron yielding 11 to 14-nucleotide-long, [beta-(32)P]ATP-labeled transcripts (D. F. Pinney and S. U. Emerson, J. Virol. 42:889-896, 1982), capping of these short RNAs was not detected. This suggests that transcripts may have to be 15 to 23 nucleotides long to be accepted as substrates by the guanyltransferase.

Base Sequence↗
PubMed1981

Vesicular stomatitis virus defective interfering particle containing a muted internal leader RNA gene.

The RNA of a unique long defective interfering particle (DI-LT2) derived from the heat-resistant strain of vesicular stomatitis virus (VSV) contains 70 nucleotides at its 3' end that are complementary to the 5' end of the VSV RNA. Following this region of terminal complementarity, there is a precise copy of the 3' end of the nondefective VSV RNA. The sequence homology between the DI-LT2 RNA and the 3' end of VSV RNA extends for at least 60 bases and probably for most of the length of the DI-LT2 RNA. The DI-LT2 particle is capable of transcription in vitro but produces only a short RNA [defective interfering (DI) particle product], which is encoded by the extreme 3' terminus of the DI RNA. Neither leader RNA nor capped VSV mRNAs are synthesized by DI-LT2, although competent templates for these are present. These data suggest that the 3'-terminal initiation is a prerequisite of the production of competent transcripts and that the sequence coding for leader RNA is not, by itself, sufficient for initiation. We propose a model for the origin of this DI particle, involving specific termination and resumption of replication, which is similar to that described previously for another class of DI particle RNAs.

Animals↗
PubMed1981

Structure and origin of a snapback defective interfering particle RNA of vesicular stomatitis virus.

The nucleotide sequence of the region which covalently links the complementary strands of the "snapback" RNA of vesicular stomatitis virus, DI011, is (Formula: see text). Both strands of the defective interfering (DI) particle RNA were complementary for their full length and were covalently linked by a single phosphate group. Because the strands were exactly the same length and complementary, template strand and daughter strand nucleocapsids generated during replication of DI 011 were undistinguishable on the basis of sequence, a property not shared by other types of DI particle RNAs. Treatment of the RNA with RNase T1 in high-ionic-strength solutions cleaved the RNA only between positions 1 and 1'. These results and the availability of the guanosine residue in position 1' to kethoxal, a reagent that specifically derivatizes guanosines of single-stranded RNA, suggest that steric constraints keep a small portion of the "turnaround" region in an open configuration. The sequence of the turnaround region was not related in any obvious way to the sequences at the 3' and 5' termini and limited the number of possible models for the origin of this type of DI particle RNA. Two models for the genesis of DI 011 RNA are discussed. We favor one in which the progenitor DI 011 RNA was generated by replication across a nascent replication fork.

Base Sequence↗
PubMed1981

Polyadenylation sites for influenza virus mRNA.

Polyadenylated transcripts of influenza virus RNA are incomplete copies of the individual genome segments, lacking sequences complementary to the 5'-terminal nucleotides of the virion RNA. By using a procedure which depends on the polyadenylic acid tail of the mRNA being encoded in part by the genome, we have determined that the common tract of uridine residues, approximately 17 to 22 nucleotides from the 5' end of each segment, is the site of polyadenylation of influenza virus mRNA.

Base Sequence↗
PubMed1981

In vivo transcription of the 5'-terminal extracistronic region of vesicular stomatitis virus RNA.

In vivo transcription and polyadenylation at the junction of the L cistron and the 5'-terminal extracistronic region of vesicular stomatitis virus RNA was investigated. Annealing of 5'32P-labeled RNA representing the 5'-terminal noncoding 77 nucleotides of vesicular stomatitis virus genomic RNA to L gene mRNA resulted in specific duplex formation. Two specific RNase T1- and RNase A resistant duplexes, 66 and 77 nucleotides long, bound to oligodeoxythymidylic acid cellulose. The specific sizes of the duplexes and their selection by oligodeoxythymidylic acid cellulose chromatography demonstrated that they were covalently linked to the polyadenylic acid tail of L gene mRNA. These data strongly suggest that the viral polymerase polyadenylates L gene mRNA in vivo by using the stretch of seven uridine residues at the end of the L cistron and that the polymerase can resume transcribing the 5'-terminal extracistronic region, resulting in a covalent linkage of the transcript to the polyadenylic acid tail of L gene mRNA.

Genes↗
PubMed1981

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.

Base Sequence↗
PubMed1981

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.

Animals↗
PubMed1981

Infection of the central nervous system produced by R1 vesicular stomatitis virus.

Previous studies of vesicular stomatitis virus infection of the central nervous system in mice have demonstrated that both temperature-sensitive mutants and defective interfering particles produce alterations from wild type disease. To examine further the role of the biologic properties of the virus, we studied the disease produced by R1-vesicular stomatitis virus, a temperature-insensitive revertant derived from the temperature-sensitive T1026 mutant. Whereas wild type produced acute encephalitis and death in 2 to 3 days in BALB/c mice, R1 produced, in contrast, a paralytic disease 5 to 6 days postinoculation and death on days 6 to 7. R1 infection is productive, although with altered kinetics of growth. No alterations of the biologic properties of the virus following intracerebral passage could be detected. The morphologic features showed a striking localization of viral replication and damage to the anterior horns of the spinal cord with sparing of the ependymal cells, unlike the situation in wild type infection. At the time of clinical onset of R1 disease, the morphologic appearances were degenerative in nature. These features correlate with the clinical localization and development of disease. In addition, morphologic evidence for trans-synaptic spread of R1 and wild type has been found. Although the delayed and altered central nervous system disease resembles aspects of delayed disease produced in other situations, it seems clear that the mechanisms operative here are unlike those previously described and may be related to a unique genomic defect in R1 or its interaction with host cell factors.

Animals↗
PubMed1980

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.

Base Sequence↗
PubMed1980

Antibody-induced modulation of rhabdovirus infection of neurons in vitro.

Dissociated neuron cultures of mice were inoculated with vesicular stomatitis virus (VSV) and subsequently fed with medium containing sufficient antiviral antibody (AB) to neutralize all free virus. In contrast to control acute neuronal infection, which lasts one to two days, AB-treated neuron cultures were maintained as long as two weeks. This protection was not obtained in infected monkey kidney cells treated with AB. Protected neuron cultures were examined with immunolabeling and EM techniques. During the first days of AB treatment, viral buds and surface antigens were often grouped in clusters instead of being diffuse on the neuronal surface. In addition, phagocytic cells often in contact with viruses released by neurons progressively engulfed aggregates of these viruses in their lysosomal systems. Dendritic processes in AB-treated cultures contained more viral antigen and ribonucleoproteins, but less assembly sites than in acutely-infected neurons. However, budding sites were frequent on the side of the post-synaptic density and some viruses seemed to enter directly into the lateral side of the presynaptic terminal to which the productive dendrite was connected. Removal of AB at this stage resulted in reactivation of viral infection in more neurons than those originally infected, but complete viral maturation and release occurred only 2 to 3 days after removal of AB. Chance of reactivation decreased with increasing length of AB treatment, and no viral antigens or budding sites were detected during the second week of AB treatment. Continuous treatment of VSV-infected neuron cultures with antiviral AB first induces (1) activation of non-neuronal cells to phagocytize clusters of viruses forming at the neuron surface, and (2) restriction of virus maturation sites mostly to the postsynaptic area where virus spreading to presynaptic endings seems favored. Prolonged treatment with AB later results in the apparent curing of the infection.

Animals↗
PubMed1980

Intervening sequence between the leader region and the nucleopcapsid gene of vesicular stomatitis virus RNA.

The base sequence at the 3' end of vesicular stomatitis virus RNA was determined by using terminal labels and chemical RNA sequencing. The leader RNA was complementary to 47 bases at the 3' terminus, whereas the nucleocapsid gene (N) began 51 nucleotides from the 3' end of the genomic RNA. The intervening bases were 3'...GAAA...5' for the Indiana serotype and 3'...GAAAA...5' for the New Jersey serotype. The complements of these bases did not appear in either the leader RNA or the N mRNA. This sequence may function as a stop signal or cleavage site during transcription. Furthermore, processing or termination at this sequence must be inhibited during the production of full-length RNA plus-sense strands (replication). We recently found similar sequences approximately 46 to 48 nucleotides from the 3' ends of several defective interfering particle RNAs where the short defective interfering particle transciption products terminate. This sequence is present also at the end of the polymerase (L) gene.

Base Sequence↗
PubMed1980

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.

Base Sequence↗
PubMed1980

Site on the vesicular stomatitis virus genome specifying polyadenylation and the end of the L gene mRNA.

The 5'-terminal nucleotide sequence from positions 50 to 130 of vesicular stomatitis virus RNA was determined indirectly by using a defective interfering particle RNA which contains covalently linked genomic minus and antigenomic plus sense RNAs. The last 18 nucleotides of the L gene coding for in the viral polymerase were identified and isolated by specific duplex formation between 5' terminally labeled oligonucleotides from a small single-stranded defective interfering particle RNA and L gene mRNA. The L gene ends at position 60 from the 5' terminus of the vesicular stomatitis genome. The data demonstrated that the first seven adenine residues in the polyadenylic acid tail of L gene mRNA may be coded for in the genome and suggested that the viral transcriptase itself may carry out polyadenylation, possibly by chattering at the uridine-rich sequence at the end of the L gene. Analysis of the 5'-terminal sequence of vesicular stomatitis virus genomic RNA revealed that it might fold into a complex secondary structure with possibly 62% of the bases paired.

Base Sequence↗
PubMed1980

Replicative RNA synthesis and nucleocapsid assembly in vesicular stomatitis virus-infected permeable cells.

A permeable-cell system has been developed to study the replication of vesicular stomatitis virus. When vesicular stomatitis virus-infected BHK cells were permeabilized by lysolecithin treatment, they incorporated nucleoside triphosphates into RNA and amino acids into proteins at nearly normal rates. The viral mRNA's synthesized appeared normal in polarity, size distribution, and polyadenylation, and all five viral proteins were synthesized. Replication of the viral genome proceeded, and full-length RNA strands were synthesized in amounts and polarities resembling those found in intact cells. These full-length RNAs associated with viral N proteins to form RNase-resistant nucleocapsids of normal buoyant density. Permeable cells appear to represent ideal hosts for studying vesicular stomatitis virus replication since they closely mimic in vivo conditions while retaining much of the experimental flexibility of current in vitro systems.

Animals↗