Continuing coevolution of virus and defective interfering particles and of viral genome sequences during undiluted passages: virus mutants exhibiting nearly complete resistance to formerly dominant defective interfering particles

We quantitatively analyzed the interference interactions between defective interfering (DI) particles and mutants of cloned vesicular stomatitis virus passaged undiluted hundreds of times in BHK-21 cells. DI particles which predominated at different times in these serial passages always interfered most strongly (and very efficiently) with virus isolated a number of passages before the isolation of the DI particles. Virus isolated at the same passage level as the predominant DI particles usually exhibited severalfold resistance to these DI particles. Virus mutants (Sdi- mutants) isolated during subsequent passages always showed increasing resistance to these DI particles, followed by decreasing resistance as new DI particles arose to predominate and exert their own selective pressures on the virus mutant population. It appears that such coevolution of virus and DI particle populations proceeds indefinitely through multiple cycles of selection of virus mutants resistant to a certain DI particle (or DI particle class), followed by mutants resistant to a newly predominant DI particle, etc. At the peak of resistance, virus mutants were isolated which were essentially completely resistant to a particular DI particle; i.e., they were several hundred thousand-fold resistant, and they formed plaques of normal size and numbers in the presence of extremely high multiplicities of the DI particle. However, they were sensitive to interference by other DI particles. Recurring population interactions of this kind can promote rapid virus evolution. Complete sequencing of the N (nucleocapsid) and NS (polymerase associated) genes of numerous Sdi- mutants collected at passage intervals showed very few changes in the NS protein, but the N gene gradually accumulated a series of stable nucleotide and amino acid substitutions, some of which correlated with extensive changes in the Sdi- phenotype. Likewise, the 5' termini (and their complementary plus-strand 3' termini) continued to accumulate extensive base substitutions which were strikingly confined to the first 47 nucleotides. We also observed addition and deletion mutations in noncoding regions of the viral genome at a level suggesting that they probably occur at a high frequency throughout the genome, but usually with lethal or debilitating consequences when they occur in coding regions.     

Utilization of homotypic and heterotypic proteins of vesicular stomatitis virus by defective interfering particle genomes for RNA replication and virion assembly: implications for the mechanism of homologous viral interference

Defective interfering (DI) particles of Indiana serotype of vesicular stomatitis virus (VSV(Ind)) are capable of interfering with the replication of both homotypic VSV(Ind) and heterotypic New Jersey serotype (VSV(NJ)) standard virus. In contrast, DI particles from VSV(NJ) do not interfere with the replication of VSV(Ind) standard virus but do interfere with VSV(NJ) replication. The differences in the interfering activities of VSV(Ind) DI particles and VSV(NJ) DI particles against heterotypic standard virus were investigated. We examined the utilization of homotypic and heterotypic VSV proteins by DI particle genomic RNAs for replication and maturation into infectious DI particles. Here we show that the RNA-nucleocapsid protein (N) complex of one serotype does not utilize the polymerase complex (P and L) of the other serotype for RNA synthesis, while DI particle genomic RNAs of both serotypes can utilize the N, P, and L proteins of either serotype without serotypic restriction but with differing efficiencies as long as all three proteins are derived from the same serotype. The genomic RNAs of VSV(Ind) DI particles assembled and matured into DI particles by using either homotypic or heterotypic viral proteins. In contrast, VSV(NJ) DI particles could assemble only with homotypic VSV(NJ) viral proteins, although the genomic RNAs of VSV(NJ) DI particles could be replicated by using heterotypic VSV(Ind) N, P, and L proteins. Thus, we concluded that both efficient RNA replication and assembly of DI particles are required for the heterotypic interference by VSV DI particles.     

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.     

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.     

Delayed formation of defective interfering particles in vesicular stomatitis virus-infected cells: kinetic studies of viral protein and RNA synthesis during autointerference.

The time course of defective interfering (DI) particle and B particle release from vesicular stomatitis virus-infected BHK-21 cells was studied at different multiplicities of defective and infective particles. Particle release was progressively delayed in cells infected with an increasing DI-to-B particle ratio. The delayed particle release during interference was found to be connected with a reduced but prolonged synthesis of viral proteins, a slower accumulation of viral proteins, and a delayed shutoff of cellular protein synthesis. The relative synthesis of M and G proteins was reduced during interference, whereas the relative synthesis of N and NS proteins was increased. On the level of genomic RNA replication, we found that DI RNA was replicated more slowly during interference than the standard genomic RNA was during acute infection. The ratio of DI particles to B particles which were released increased throughout the infectious cycle. At a given time in the infectious cycle, this ratio was independent of the multiplicity of infecting DI and B particles. On the basis of the kinetic studies, we argue that cells infected with higher amounts of DI particles compared with B particles synthesize a higher DI-to-B particle ratio and release these progeny particles later than cells infected with a low DI-to-B particle ratio.     

Vesicular stomatitis virus defective interfering particles can contain extensive genomic sequence rearrangements and base substitutions

We sequenced the 5' and 3' RNA termini of 16 defective interfering (DI) particles of vesicular stomatitis virus (VSV) isolated at intervals from persistent infections and from a series of undiluted lytic passages. All DI RNAs exhibited complementary termini, but sequences internal to these termini were extensively rearranged in a variety of ways. Despite extensive rearrangement, these internal sequences (in addition to the termini) apparently are important for DI particle interference properties. Some of these DI particles are derived from multiple intrastrand and interstrand recombination events, and the generation of each can be explained by current replicase error models. During viral evolution in persistent and acute infections, DI particles with specific termini base substitutions are selected. One DI particle exhibits a remarkable clustering of specific A----G (and complementary U----C) substitutions, apparently as a result of repetitive misincorporations by an error-prone viral polymerase complex.     

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.     

Segment-specific noncoding sequences of the influenza virus genome RNA are involved in the specific competition between defective interfering RNA and its progenitor RNA segment at the virion assembly step.

The generation of influenza A virus defective interfering (DI) particles was studied by using an NS2 mutant which produces, in a single cycle of virus replication, a large amount of DI particles lacking the PA polymerase gene. The decrease in PA gene replication has been shown to occur primarily at the cRNA synthesis step, with preferential amplification of PA DI RNA species present in a marginal amount in the virus stock. In addition, at the assembly step the PA DI RNAs were preferentially incorporated into virions, resulting in selective reduction in the packaging of the PA gene into virions. Similarly, in cells dually infected with the NS2 mutant and wild-type viruses, packaging of the wild-type PA gene was also greatly suppressed. In contrast, incorporation of other RNA segments, i.e., the PB2 and NS genes, was not affected, suggesting that the PA DI RNAs competed only with the PA gene in a segment-specific manner. Experiments involving rescue of recombinant chloramphenicol acetyltransferase (CAT) RNA flanked by the noncoding regions of the PA (PA/CAT RNA) and PB2 (PB2/CAT RNA) genes into viral particles showed that only PA/CAT RNA was not rescued by infection with the NS2 mutant virus containing the PA DI RNAs. However, recombinant PA/CAT RNA in which either the 3' or 5' noncoding region was replaced with that of the PB2 gene was rescued by the NS2 mutant. These results suggest that the noncoding regions of the PA gene are responsible for the competition with PA DI RNA species at the virus assembly step and that coexistence of the both noncoding regions would be a prerequisite for this phenomenon. Decreased packaging of the progenitor RNA by the DI RNA, in addition to the suppression of cRNA synthesis, is likely involved in the production of DI particles.     

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.     

Vesicular stomatitis virus N and NS proteins form multiple complexes.

The vesicular stomatitis virus nucleocapsid protein, N, associated specifically with the viral phosphoprotein, NS, in an in vitro system which supported vesicular stomatitis virus RNA replication. Essentially all the N protein was found complexed with NS. In addition, multiple forms of the N-NS complex were detected which differed in their sedimentation properties and ratios of N to NS.     

The nucleotide sequence of the 5'' terminus of vesicular stomatitis virus RNA.

We have determined the nucleotide sequence for the first 50 nucleotides at the 5' terminus of vesicular stomatitis virus (VSV) genome RNA. This sequence is identical to that of the in vitro RNA polymerase product synthesized by defective interfering (DI) particles of VSV. These results confirm previous conclusions rengarding DI and standard viral terminal sequences based on hybridization studies and earlier sequencing of the DI polymerase product RNA.     

Properties of DI particle resistant mutants of vesicular stomatitis virus isolated from persistent infections and from undiluted passages

We describe an assay procedure to quantitate relative DI resistance of a variety of DI particle resistant (Sdi^?) mutants of vesicular stomatitis virus (VSV). We show that numerous diverse Sdi^? mutants of VSV are selected continuously in a stepwise manner during persistent infections, and also during serial undiluted lytic passages initiated with cloned virus. Concurrently with the successive appearance and disappearance of different Sdi^? mutants of infectious VSV, new DI particle types with altered interference properites also appear and disappear, resulting in rapid “coevolution” of virus and DI particle populations. Complementation tests with Sdi^? mutants indicate that mutations in at least two different virus factors (presumably associated with replication-encapsidation) can give rise to Sdi^? mutants. Interference studies with chimeric DI particles indicate that DI particle template RNA rather than DI particle protein determines the interference properties of DI particles interacting with Sdi^? and Sdi⁺ mutants of helper virus.     

Defective Interfering Particles of Vesicular Stomatitis Virus: Functions of the Genomic Termini

The functions of thecis-acting sequence elements at the termini of the negative strand RNA virus vesicular stomatitis virus and its defective-interfering particles were evaluated. Either genomic terminus could signal replication but increasing the complementarity of the termini enhanced replication irrespective of whether the termini consisted of the 3′ leader and its complement or the 5′ trailer and its complement. The 5′ trailer region contains an essentialcis-acting requirement for assembly of RNPs into infectious particles. These findings explain why the majority of DI RNAs are of the 5′ copy-back class: RNAs with complementary termini from either end have a replicative advantage, but only 5′ copy-back RNAs contain the signal for assembly into particles.     

Defective Particles in BHK Cells Infected with Temperature-Sensitive Mutants of Vesicular Stomatitis Virus

Defective particles were the major product after undiluted passage of certain temperature-sensitive (ts) mutants of the Indiana C strain of vesicular stomatitis virus in BHK-21 cells at the permissive temperature (31 C). Essentially homogeneous preparations of defective particles were obtained with the wild-type and individual ts mutants. The defective particles associated with some of the ts mutants, however, were morphologically and physically distinguishable from wild type and from each other. All varieties of defective particle interfered with the multiplication of mutant and wild-type virus at the permissive temperature at early times of infection but failed to complement virions of different complementation groups at the restrictive temperature (39 C) at any time during infection.