Analysis of the recombination event generating a vesicular stomatitis virus deletion defective interfering particle.

cDNA clones of different portions of the L cistron and 5'-terminal region of the vesicular stomatitis virus genome have been prepared and used to identify the exact site of the deletion in the defective interfering particle, DI-LT. The deletion extends from nucleotide 251 from the beginning of the L gene to a position 342 nucleotides from the end of the genome. The nucleotide sequences flanking the deletion site, as well as those at the ends of the deleted segment, did not contain any obvious vesicular stomatitis virus initiation or termination signals as had been found near the recombination sites in other defective interfering particle RNAs. The results best fit a model for the origin of this type of defective interfering particle in which the polymerase interrupts its synthesis and moves with its nascent daughter strand to a new position on the template and resumes synthesis there, further extending the nascent strand. Neither the interruption nor the resumption of synthesis appears to be in response to the template nucleotide sequence. The sequences of two partial L cistron clones also reveal open reading frames that code for amino acid sequences likely to be the amino and carboxy termini of the L protein.     

Interference among defective interfering particles of vesicular stomatitis virus

Three defective interfering (DI) particles of vesicular stomatitis virus (VSV), all derived from the same parental standard San Juan strain (Indiana serotype), were used in various combinations to infect cells together with the parental virus. The replication of their RNA genomes in the presence of other competing genomes was described by the hierarchical sequence: DI 0.52 particles greater than DI 0.45 particles less than or equal to DI-T particles greater than standard VSV. The advantage of one DI particle over another was not due simply to multiplicity effects nor to the irreversible occupation of limited cellular sites. Interference, however, did correlate with a change in the ratio of plus and minus RNA templates that accumulated intracellularly and with the presence of new sequences at the 3' end of the DI genomes. DI 0.52 particles contained significantly more nucleotides at the 3' end that were complementary to those at the 5' end of its RNA than did DI-T or DI 0.45 particles. The first 45 nucleotides at the 3' ends of all of the DI RNAs were identical. VSV and its DI particles can be separated into three classes, depending on their terminal RNA sequences. These sequences suggest two mechanisms, one based on the affinity of polymerase binding and the other on the affinity of N-protein binding, that may account for interference by DI particles against standard VSV and among DI particles themselves.     

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.     

Replication of vesicular stomatitis virus defective interfering particle RNA in vitro: transition from synthesis of defective interfering leader RNA to synthesis of full-length defective interfering RNA.

The replication of the RNA of vesicular stomatitis virus (VSV) defective interfering (DI) particles was established in a defined cell-free system. The transition from synthesis of only the DI-leader RNA to replication of the full-length DI RNA was effected in the system by newly synthesized VSV proteins and occurred in the absence of VSV helper virus. Both positive- and negative-polarity full-length DI RNA were synthesized. Furthermore, the products of RNA replication associated with newly synthesized viral proteins to form complexes that were indistinguishable from authentic DI particle nucleocapsids on the basis of buoyant density and resistance to ribonuclease digestion. The DI-leader RNA did not form ribonuclease-resistant structures. We conclude that this in vitro system successfully executes many of the reactions of VSV DI particle replication and assembly.     

Comparison of vesicular stomatitis virus defective interfering particle synthesis in chick embryo and L cells.

A comparison of the ability of vesicular stomatitis virus (VSV) to generate and replicate defective interfering (DI) particles in primary chick embryo (CE) and mouse L cells was investigated as a means of analyzing host control over DI-particle synthesis and interfering capacity. Serial undiluted passage of VSV in CE and L cells indicate that VSV-DI particles are generated and (or) replicate with greater efficiency in CE than in L cells. When DI particles accumulate in L cells, they are able to interfere with infectious particle replication. The DI particles from CE cells interfered to the same extent with infectious particle replication in both CE and L cells. L cells, therefore, are not considered 'low-interference' hosts in which DI particles are produced and do not interfere with infectious virus replication, but rather hosts which restrict the production of DI particles.     

Host function-dependent induction of defective interfering particles of vesicular stomatitis virus.

Suppression of host cell function by treatment with actinomycin D prior to infection prevented the induction of defective interfering particles of vesicular stomatitis virus, which had been cloned and propagated in cell pretreated with actinomycin D. Replication of defective interfering particles already present in an infecting virus stock, however, was not affected by pretreatment of cells with actinomycin D. Thus, the induction, but not the replication, of defective interfering particles appears to be a host cell function-dependent phenomenon. The implications of this phenomenon for host defense mechanisms against virus infections are discussed.     

Proposal for a uniform nomenclature for defective interfering viruses of vesicular stomatitis virus.

Defective interfering particles of vesicular stomatitis virus have been named according to their parental derivation and to their genomic length and physical properties. This suggested uniform nomenclature can be adapted for other virus systems.     

Structure and origin of a novel class of defective interfering particle of vesicular stomatitis virus

The genome structure and terminal sequences of a 'copyback' defective interfering (DI) particle ST1, and a novel complexly rearranged 'snapback' DI particle ST2 of vesicular stomatitis virus have been determined. The ST1 DI genome RNA possesses 54 base long inverted complementary termini, the 5' end of which is homologous to the standard virus genome 5' end. Following this region of inverted complementarity the DI RNA 5' end continues to be homologous to standard virus RNA 5' sequences, whereas the 3' end diverges into sequences within the virus L gene internal sequences. ST2 DI genome RNA does not contain colinear covalently linked plus and minus sense RNA copies of the standard infectious virus RNA 5' terminus as predicted from the prototype snapback DI structure, but instead appears to be a hairpin copy of the ST1 DI RNA genome. This is the first evidence suggesting that DI particles may be generated from RNA templates other than the standard virus RNA. Generation models and the implications of these findings for RNA virus evolution are discussed.     

Inhibition of pseudorabies virus replication by vesicular stomatitis virus. II Activity of defective interfering particles.

Purified defective interfering (DI) particles of vesicular stomatitis virus (VSV) inhibit the replication of a heterologous virus, pseudorabies virus (PSR), in hamster (BHK-21) and rabbit (RC-60) cell lines. In contrast to infectious B particles of VSV, UV irradiation of DI particles does not reduce their ability to inhibit PSR replication. However, UV irradiation progressively reduces the ability of DI particles to cause homologous interference with B particle replication. Pretreatment with interferon does not affect the ability of DI particles to inhibit PSR replication in a rabbit cell line (RC-60) in which RNA, but not DNA, viruses are sensitive to the action of interferon. Under similar conditions of interferon pretreatment, the inhibition of PSR by B particles is blocked. These data suggest that de novo VSV RNA or protein synthesis is not required for the inhibition of PSR replication by DI particles. DI particles that inhibit PSR replication also inhibit host RNA and protein synthesis in BHK-21 and RC-60 cells. Based on the results described and data in the literature, it is proposed that the same component of VSV B and DI particles is responsible for most, if not all, of the inhibitory activities of VSV, except homologous interference.     

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.     

Isolation of vesicular stomatitis virus defective interfering genomes with different amounts of 5''-terminal complementarity.

I isolated at least 30 different vesicular stomatitis virus defective interfering (DI) genomes, distinguished by chain length, by five independent undiluted passages of a repeatedly cloned virus plaque. Labeling of the 3' hydroxyl ends of these DI genomes and RNase digestion studies demonstrated that the ends of these DI genomes were terminally complementary to different extents (approximately 46 to 200 nucleotides). Mapping studies showed that the complementary ends of all of the DI genomes were derived from the 5' ends of the nondefective minus-strand genome. Regardless of the extent of terminal complementarity, all of the DI genomes synthesized the same 46-nucleotide minus-strand leader RNA.