Defective interfering particles of poliovirus. 3. Interference and enrichment

Interference with standard poliovirus growth resulting from co-infection of cells with standard virus and defective interfering particles has been investigated. At all time following infection, co-infected cells produced less standard progeny than cells infected only by standard virus. The total yield of physical particles and the percentage of standard virus among these particles was a linear function of the percentage of standard virus in the inoculum. The actual yield of standard virus thus varied as the square of the percentage of standard virus in the inoculum. The extent of interference could also be controlled by varying the time interval between initial infection of cells by one type of particle and superinfection by the other.Identical amounts of viral RNA and virus-specific polyribosomes are formed in co-infected or singly infected cells. Interference apparently results from the partitioning of these limited synthetic capacities between standard and defective interfering-specific RNA and protein synthesis. Standard and DI RNA appear to serve equally well as messenger RNAs because standard and DI-specific viral proteins are synthesized in ratios proportional to the ratio of standard to DI particles in the inoculum. Only standard RNA can direct the formation of capsid protein, so co-infected cells contain reduced amounts of the virion protein precursor, the procapsid. Standard and DI RNA are encapsidated with approximately equal efficiency. Thus interference results from equal participation in the intracellular events of the infection cycle by both types of particles.The progeny yield from co-infected cells was always enriched about 5 to 8% in DI particles. Progeny were produced in the enriched ratio throughout the infection cycle.     

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.     

Homologous interference mediated by defective interfering influenza virus derived from a temperature-sensitive mutant of influenza virus.

A temperature-sensitive group II mutant of influenza virus, ts-52, with a presumed defect in viral RNA synthesis, readily produced von Magnus-type defective interfering virus (DI virus) when passed serially (four times) at high multiplicity in MDBK cells. The defective virus (ts-52 DI virus) had a high hemagglutinin and a low infectivity titer, and strongly interfered with the replication of standard infectious viruses (both ts-52 and wild-type ts+) in co-infected cells. Progeny virus particles produced by co-infection of DI virus and infectious virus were also defective and also had low infectivity, high hemagglutinating activity, and a strong interfering property. Infectious viruses ts+ and ts-52 were indistinguishable from ts-52 DI viruses by sucrose velocity or density gradient analysis. Additionally, these viruses all possessed similar morphology. However, when the RNA of DI viruses was analyzed by use of polyacrylamide gels containing 6 M urea, there was a reduction in the amount of large RNA species (V1 to V4), and a number of new smaller RNA species (D1 to D6) with molecular weights ranging from 2.9 X 10(5) to 1.05 X 10(5) appeared. Since these smaller RNA species (D1 to D6) were absent in some clones of infectious viruses, but were consistently associated with DI viruses and increased during undiluted passages and during co-infection of ts-52 with DI virus, they appeared to be a characteristic of DI viruses. Additionally, the UV target size of interfering activity and infectivity of DI virus indicated that interfering activity was 40 times more resistant to UV irradiation than was infectivity, further implicating small RNA molecules in interference. Our data suggest that the loss of infectivity observed among DI viruses may be due to nonspecific loss of a viral RNA segment(s), and the interfering property of DI viruses may be due to interfering RNA segments (DIRNA, D1 to D6). ts-52 DI virus interfered with the replication of standard virus (ts+) at both permissive (34 degrees C) and nonpermissive temperatures. The infectivity of the progeny virus was reduced to 0.2% for ts+ and 0.05% for ts-52 virus without a reduction in hemagglutinin titer. Interference was dependent on the concentration of DI virus. A particle ratio of 1 between DI virus (0.001 PFU/cell) and infectious virus (1.0 PFU/cell) produced a maximal amount of interference. Infectious virus yield was reduced 99.9% without any reduction of the yield of DI viruses Interference was also dependent on the time of addition of DI virus. Interference was most effective within the first 3 h of infection by infectious virus, indicating interference with an early function during viral replication.     

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.     

Replication-defective viruses modulate immune responses.

By immunizing inbred mice with purified replication-competent, defective virus particles, or an admixture of the two, differential effects on the cellular immune system have been uncovered. Defective virus, exemplified by the vesicular stomatitis virus (VSV) defective interfering particle (DI 0.33), induced in BALB/c mice low levels of proliferating, IL-2 secreting, and cytolytic Ag-specific T lymphocytes. This was not caused by a dominant suppressor cell response, or by a failure to stimulate lymphokine-secreting cells, but appeared to reflect a reduced efficiency of priming as compared with standard virus. Mice primed with a mixture of wt and DI virus showed reduced proliferation compared with mice primed with wt virus. When histocompatible target cells were sensitized by pure DI particles, they were neither recognized nor lysed by CD8+ CTL. Cells co-infected with wt and DI particles were not as readily lysed by CD8+ CTL as cells infected by VSV alone. The extent of this reduction was dependent on the concentration of DI particles. This suggests that DI particles may have prevented the proper presentation of endogenously synthesized Ag for recognition by CD8+ CTL. Metabolic labeling studies indicated that the presence of DI particles suppressed the synthesis of viral proteins in dually infected cells. However, CD4+ T lymphocyte clones recognized and efficiently lysed histocompatible Ia+ cells infected with DI particles alone or co-infected with replication-competent and defective virus.     

Synthesis of virus-specific polypeptides and genomic RNA during the replicative cycle of Pichinde virus

A stock of plaque-purified Pichinde virus, prepared under conditions designed to limit the amounts of defective interfering virus, was used to infect BHK cells. At daily intervals after infection, cells were examined for infectious and radiolabeled virus particle production and for the synthesis of virus-specific polypeptides. Quantitative comparisons were also made of the concentrations of genomic Pichinde virus L and S RNAs in the cytoplasm of infected cells on different days after infection. Our results showed that virus particle production, rates of protein synthesis, and the intracellular levels of viral genomic RNAs all increased and decreased with similar kinetics, and that this regulation was independent of the cell growth cycle. We were unable to relate these changes in viral macromolecule and virus production to the appearance of readily identifiable defective interfering particles. Our findings suggest that regulation of virus replication early during the replicative cycle of Pichinde virus may not be dependent upon the generation of defective interfering virus.     

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.     

Defective interfering viral particles in acute dengue infections

While much of the genetic variation in RNA viruses arises because of the error-prone nature of their RNA-dependent RNA polymerases, much larger changes may occur as a result of recombination. An extreme example of genetic change is found in defective interfering (DI) viral particles, where large sections of the genome of a parental virus have been deleted and the residual sub-genome fragment is replicated by complementation by co-infecting functional viruses. While most reports of DI particles have referred to studies in vitro, there is some evidence for the presence of DI particles in chronic viral infections in vivo. In this study, short fragments of dengue virus (DENV) RNA containing only key regulatory elements at the 3' and 5' ends of the genome were recovered from the sera of patients infected with any of the four DENV serotypes. Identical RNA fragments were detected in the supernatant from cultures of Aedes mosquito cells that were infected by the addition of sera from dengue patients, suggesting that the sub-genomic RNA might be transmitted between human and mosquito hosts in defective interfering (DI) viral particles. In vitro transcribed sub-genomic RNA corresponding to that detected in vivo could be packaged in virus like particles in the presence of wild type virus and transmitted for at least three passages in cell culture. DENV preparations enriched for these putative DI particles reduced the yield of wild type dengue virus following co-infections of C6-36 cells. This is the first report of DI particles in an acute arboviral infection in nature. The internal genomic deletions described here are the most extensive defects observed in DENV and may be part of a much broader disease attenuating process that is mediated by defective viruses.     

Defective interfering particles of poliovirus. II. Nature of the defect

Poliovirus defective, interfering particles in which about 15% of the standard viral RNA is deleted have been described (Cole et al., 1971). Stocks of DI³ particles more than 99% free of standard poliovirus were prepared by centrifugation of mixed preparations in CsCl gradients. Using purified DI particles, it was found that DI particles can carry out most of the standard poliovirus functions including inhibition of cellular macromolecular synthesis, production of viral RNA and production of virus-specific protein. Neither the kinetics nor extent of viral RNA or protein synthesis differed between DI particle-infected cells and standard virus-infected cells.Newly made virions, capsid proteins, and the capsid protein precursor (NCVP 1) were totally absent in DI particle-infected cells. All of the other viral proteins were present. DI-infected cells briefly labeled with amino acids also contained a new polypeptide, DI-P, which was apparently the residual fragment of NCVP 1 encoded by the DI genome. It was very unstable, being rapidly degraded to acid-soluble fragments. When the cleavage of viral proteins was inhibited with amino acid analogs, precursors of the viral proteins were generated. Those precursors which should have contained NCVP 1 had molecular weights 30,000 to 40,000 daltons lower in DI-infected cells than in standard virus-infected cells. This is the amount of protein encoded by 15% of the standard poliovirus genome which is the per cent of the standard RNA sequence not represented in DI RNA.Poliovirus DI particles therefore appear to be deletion mutants lacking RNA encoding about one-third of the capsid protein precursor. Whether the deletion is internal or terminal remains to be determined.     

Defective Interfering Particles of Poliovirus IV. Mechanisms of Enrichment

Infection of HeLa cells by mixtures of standard poliovirus and defective, interfering (DI) poliovirus particles leads to a higher ratio of DI particles in the progeny than in the inoculum. The extent of this enrichment could be varied by various manipulations of the co-infected cells. At any time during the infection cycle, virions made within short times after addition of radioactive uridine were hyperenriched in DI particles; this transient hyperenrichment fell to the equilibrium enrichment level within 45 min after uridine addition. A shift of the temperature of infection from 37 to 31 C also led to a hyperenrichment of DI particles and pulse-labeling revealed a superimposed transient hyperenrichment. By contrast, cells continuously infected at 31 C showed a severe decrement in DI particles apparently because poliovirus DI particles behave as cold-sensitive mutants for RNA synthesis. Cycloheximide treatment early in the infection cycle also led to hyperenrichment. Study of the cycloheximide effect showed that the drug acted as if to change the input ratio of standard to DI particles. These effects on enrichment can be explained as aspects of two different phenomena: enrichment due to preferential DI RNA synthesis and enrichment due to preferential encapsidation of DI RNA. Both mechanisms probably play a role in the normal level of enrichment.     

In vitro construction of poliovirus defective interfering particles.

To construct poliovirus defective interfering (DI) particles in vitro, we synthesized an RNA from a cloned poliovirus cDNA, pSM1(T7)1, which carried a deletion in the genome region corresponding to nucleotide positions 1663 to 2478 encoding viral capsid proteins, by using bacteriophage T7 RNA polymerase. The RNA was designed to retain the correct reading frame in nucleotide sequence downstream of the deletion. HeLa S3 monolayer cells were transfected with the deletion RNA and then superinfected with standard virus as a helper. The DI RNA was observed in the infected cells after three passages at high multiplicity of infection. The sequence analysis of RNA extracted from the purified DI particle clearly showed that this DI RNA had the same deletion in size and location as that in the RNA used for the transfection. Thus, we succeeded in construction of a poliovirus DI particle in vitro. To gain insight into the mechanism for DI generation, we constructed poliovirus cDNAs pSM1(T7)1a and pSM1(T7)1b that, in addition to the same deletion as that in pSM1(T7)1, had insertion sequences of 4 bases and 12 bases, respectively, at the corresponding nucleotide position, 2978. The RNA transcribed from pSM1(T7)1a was not a template for synthesis of poliovirus nonstructural proteins and therefore was inactive as an RNA replicon. On the other hand, the RNA from pSM1(T7)1b replicated properly in the transfected cells. Superinfection of the transfected cells with standard virus resulted in production of DI particles derived from pSM1(T7)1b and not from pSM1(T7)1a. These observations indicate that deletion RNAs that are inactive replicons have little or no possibility of being genomes of DI particles suggesting the existence of a nonstructural protein(s) that has an inclination to function as a cis-acting protein(s). The method described here will provide a useful technique to investigate genetic information essential for poliovirus replication.     

Replication of nodamura virus after transfection of viral RNA into mammalian cells in culture.

Nodamura virus (NOV) was purified from the hind limbs of infected suckling mice and used as a source of the two genomic RNAs of the virus, RNA 1 and RNA 2. Upon transfection of the viral RNAs into baby hamster kidney (BHK21) cells in culture, vigorous RNA replication ensued and single-stranded RNAs 1 and 2 accumulated to reach an abundance which approximated that of the cellular rRNAs. Transient synthesis of a small subgenomic RNA (RNA 3) was also observed, and double-stranded versions of RNAs 1, 2, and 3 were detected. Three major viral proteins were synthesized in transfected cells. Protein A (about 115 kDa) and protein B (about 15 kDa) were made transiently at early times after transfection, whereas a large amount of protein alpha (43 kDa), the precursor to the two viral coat proteins, was made continuously starting later in the infectious cycle. When very low concentrations of viral RNAs were used for transfection, preferential replication of RNA 1 occurred. This result was attributed to segregation of the transfected viral RNAs to separate cells in culture and the subsequent replication and amplification of RNA 1 in cells that had received no RNA 2. Accordingly, multiple passages of the viral RNAs by transfection at the limit dilution resulted in the purification of RNA 1 free of RNA 2 and demonstrated that RNA 1 was capable of prolonged autonomous replication which was also accompanied by the continuous synthesis of RNA 3. In cells transfected with RNA 1 alone, protein alpha was not synthesized and proteins A and B were made continuously. Electron microscopic analysis of BHK21 cells 24 h after transfection with NOV RNAs 1 and 2 showed that large numbers of virus particles accumulated in the cytoplasm and formed paracrystalline arrays in some regions. Whole NOV purified from transfected BHK21 cells was infectious for suckling mice and had an electrophoretic mobility that was similar but not identical to that of NOV purified from infected mouse muscle. The high yield of NOV, its simple genetic composition, and its unusual genome strategy make this virus an attractive system for the study of viral RNA replication in animal cells.     

Defective interfering virus particles modulate virulence.

To determine whether defective interfering (DI) particles modulate virulence by initiating a cyclic pattern of virus growth in vivo, adult mice were infected with vesicular stomatitis virus (VSV), both with and without DI particles. A total of 184 mice divided into groups were inoculated intranasally. A majority of mice inoculated only with standard VSV developed paralysis, most of them between days 7 and 9. The addition of DI particles altered the development of paralysis in several ways. When there was significant protection, a few still became paralyzed on days 7 and 9. When overall mortality was unaffected or even slightly increased, the majority of mice became paralyzed between days 7 and 9 as well. Protection could not be predicted based on a single ratio of standard VSV to DI particles or on the absolute amount of DI particles inoculated. Infectious virus recovered from mouse brains at the time of paralysis and incipient death showed considerable variation, although the titer in a majority of the animals was between 10(5) and 10(7) PFU/ml. When the brains of these paralyzed mice were examined for hybridizable VSV RNA, the detection of standard VSV RNA correlated well with infectivity. The amount of DI RNA in the coinfected mice was more variable and independent of the amount of 40S RNA, although DI RNA was usually found when standard RNA was present. Survivors examined between days 14 and 21 did not contain infectious virus or any detectable viral RNA in their brains. Because these results were consistent with the hypothesis of viral cycling in vivo, rather than a gradual accumulation of total infectious virus, mice were coinfected with 10(8) PFU of standard VSV and 10(5) PFU equivalents of DI particles and sacrificed daily thereafter, irrespective of whether they developed paralysis. Infectivity measurements indicated a reproducible cycling pattern of VSV in the mouse brains with a periodicity of about 5 days. This cycling and the detection of DI RNA in brains several days after intranasal inoculation suggest that there is a dynamic continuous interaction between standard VSV and its DI particle beyond the initial site of replication as the virus population spreads into the host animal. Such cycling of virus production before the full development of specific immune responses from the host may have important implications for viral diagnostics and disease transmission.     

Complementation of a poliovirus defective genome by a recombinant vaccinia virus which provides poliovirus P1 capsid precursor in trans.

Defective interfering (DI) RNA genomes of poliovirus which contain in-frame deletions in the P1 capsid protein-encoding region have been described. DI genomes are capable of replication and can be encapsidated by capsid proteins provided in trans from wild-type poliovirus. In this report, we demonstrate that a previously described poliovirus DI genome (K. Hagino-Yamagishi and A. Nomoto, J. Virol. 63:5386-5392, 1989) can be complemented by a recombinant vaccinia virus, VVP1 (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 65:2088-2092, 1991), which expresses the poliovirus capsid precursor polyprotein, P1. Stocks of defective polioviruses were generated by transfecting in vitro-transcribed defective genome RNA derived from plasmid pSM1(T7)1 into HeLa cells infected with VVP1 and were maintained by serial passage in the presence of VVP1. Encapsidation of the defective poliovirus genome was demonstrated by characterizing poliovirus-specific protein expression in cells infected with preparations of defective poliovirus and by Northern (RNA) blot analysis of poliovirus-specific RNA incorporated into defective poliovirus particles. Cells infected with preparations of defective poliovirus expressed poliovirus protein 3CD but did not express capsid proteins derived from a full-length P1 precursor. Poliovirus-specific RNA encapsidated in viral particles generated in cells coinfected with VVP1 and defective poliovirus migrated slightly faster on formaldehyde-agarose gels than wild-type poliovirus RNA, demonstrating maintenance of the genomic deletion. By metabolic radiolabeling with [35S]methionine-cysteine, the defective poliovirus particles were shown to contain appropriate mature-virion proteins. This is the first report of the generation of a pure population of defective polioviruses free of contaminating wild-type poliovirus. We demonstrate the use of this recombinant vaccinia virus-defective poliovirus genome complementation system for studying the effects of a defined mutation in the P1 capsid precursor on virus assembly. Following removal of residual VVP1 from defective poliovirus preparations, processing and assembly of poliovirus capsid proteins derived from a nonmyristylated P1 precursor expressed by a recombinant vaccinia virus, VVP1 myr- (D. C. Ansardi, D. C. Porter, and C. D. Morrow, J. Virol. 66:4556-4563, 1992), in cells coinfected with defective poliovirus were analyzed. Capsid proteins generated from nonmyristylated P1 did not assemble detectable levels of mature virions but did assemble, at low levels, into empty capsids.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.