Defective interfering influenza viruses and host cells: establishment and maintenance of persistent influenza virus infection in MDBK and HeLa cells.

WSN (H0N1) influenza virus upon undiluted passages in different species of cells, namely, bovine kidney (MDBK), chicken embryo (CEF), and HeLa cells, produced a varying amount of defective interfering (DI) virus which correlated well with the ability of the species of cell to produce infectious virus. However, the nature of the influenza DI viral RNA produced from a single clonal stock was essentially identical in all three cells types, suggesting that these cells do not exert a great selective pressure in the amplification of specific DI viral RNAs either at early or late passages. DI viruses produced from one subtype (H0N1) could interfere with the replication of infectious viruses belonging to other subtypes (H1N1, H3N2). DI viral RNAs could also replicate with the helper function of other subtype viruses. The persistent infection of MDBK and HeLa cells could be initiated by coinfecting cells with both temperature-sensitive mutants (ts-) and DI influenza viruses. Persistently infected cultures cultures at early passages (up to passage 7) showed a cyclical pattern of cell lysis and virus production (crisis), whereas, at later passages (after passage 20), they produced little or no virus and were resistant to infection by homologous virus but not by heterologous virus. The majority of persistently infected cells, however, contained the complete viral genome since they expressed viral antigens and produced infectious centers. Selection of a slow-growing temperature-sensitive variant rather than the presence of DI virus or interferon appears to be critical in maintaining persistent influenza infection in these 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.     

Generation of defective interfering particles of Semliki Forest virus in a clone of Aedes albopictus (mosquito) cells.

Serial undiluted passage of Semliki Forest virus in a clone of Aedes albopictus cells resulted in a marked decrease in infectious virus yields due to the generation and accumulation of defective interfering particles. Virus from the third passage had a high particle/infectivity ratio and interfered specifically with homologous but not heterologous standard virus replication. Two RNA species of molecular weights 0.78 X 10(6) and 0.61 X 10(6) were the major RNA components of purified passage 4 virus. These RNA species were also the predominant virus RNA species detected in cells infected with passage 3 virus. Synthesis of standard virus RNA and virus-specified protein was much reduced in passage 3 virus-infected cells. Interference with standard virus replication and the synthesis of large amounts of defective interfering RNA were also observed in chicken embryo cells infected with passage 3 virus from mosquito cells.     

Generation of a purely clonal defective interfering influenza virus

Defective interfering (DI) influenza viruses carry a large deletion in a gene segment that interferes with the replication of infectious virus; thus, such viruses have potential for antiviral therapy. However, because DI viruses cannot replicate autonomously without the aid of an infectious helper virus, clonal DI virus stocks that are not contaminated with helper virus have not yet been generated. To overcome this problem, we used reverse genetics to generate a clonal DI virus with a PB2 DI gene, amplified the clonal DI virus using a cell line stably expressing the PB2 protein, and confirmed its ability to interfere with infectious virus replication in vitro. Thus, our approach is suitable for obtaining purely clonal DI viruses, will contribute to the understanding of DI virus interference mechanisms and can be used to develop DI virus‐based antivirals.     

Defective Influenza Viral Ribonucleoproteins Cause Interference

Ribonucleoproteins (RNPs) isolated from infectious and defective interfering (DI) influenza virus (WSN) contained three major RNP peaks when analyzed in a glycerol gradient. Peak I RNP was predominant in infectious virus but was greatly reduced in DI virus preparations. Conversely, peak III RNP was elevated in DI virus, suggesting a large increase in DI RNA in this fraction. Labeled [(32)P]RNA was isolated from each RNP region and analyzed by electrophoresis on polyacrylamide gels. Peak I RNP contained primarily the polymerase and some HA genes, peak II contained some HA gene but mostly the NP and NA genes, and peak III contained the M and NS genes. In addition, peak III RNP from DI virus also contained the characteristic DI RNA segments. Interference activity of RNP fractions isolated from infectious and DI virus was tested using infectious center reduction assay. RNP peaks (I, II, and III) from infectious virus did not show any interference activity, whereas the peak III DI RNP caused a reduction in the number of infectious centers as compared to controls. Similar interference was not demonstrable with peak I RNP of DI virus nor with any RNP fractions from infectious virus alone. The interference activity of RNP fractions was RNase sensitive, suggesting that the DI RNA contained in DI RNPs was the interfering agent, and dilution experiments supported the conclusion that a single DI RNP could cause interference. The interfering RNPs were heterogeneous, and the majority migrated slower than viral RNPs containing M and NS genes. These results suggest that DI RNP (or DI RNA) is also responsible for interference in segmented, negative-stranded viruses.     

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.     

A replication silencer element in a plus-strand RNA virus

Replication represents a key step in the infectious cycles of RNA viruses. Here we describe a regulatory RNA element, termed replication silencer, that can down-regulate complementary RNA synthesis of a positive-strand RNA virus via an RNA-RNA interaction. This interaction occurs between the 5-nucleotide-long, internally positioned replication silencer and the extreme 3'-terminus of the viral RNA comprising part of the minimal minus-strand initiation promoter. Analysis of RNA synthesis in vitro, using model defective interfering (DI) RNA templates of tomato bushy stunt virus and a partially purified, RNA-dependent RNA polymerase preparation from tombusvirus-infected plants, revealed that this interaction inhibits minus-strand synthesis 7-fold. This functional interaction was supported further by: (i) RNA structure probing; (ii) phylogenetic analysis; (iii) inhibition of activity by short complementary DNAs; and (iv) compensatory mutational analysis. The silencer was found to be essential for accumulation of DI RNAs in protoplasts, indicating that it serves an important regulatory role(s) in vivo. Because similar silencer-promoter interactions are also predicted in other virus genera, this type of RNA-based regulatory mechanism may represent a widely utilized strategy for modulating replication.     

Rubella virus capsid protein modulates viral genome replication and virus infectivity

The structural proteins (SP) of the Togaviridae can be deleted in defective interfering RNAs. The dispensability of viral SP has allowed construction of noninfectious viral expression vectors and replicons from viruses of the Alphavirus and Rubivirus genera. Nevertheless, in this study, we found that the SP of rubella virus (RUB) could enhance expression of reporter genes from RUB replicons in trans. SP enhancement required capsid protein (CP) expression and was not due to RNA-RNA recombination. Accumulation of minus- and plus-strand RNAs from replicons was observed in the presence of SP, suggesting that SP specifically affects RNA synthesis. By using replicons containing an antibiotic resistance gene, we found 2- to 50-fold increases in the number of cells surviving selection in the presence of SP. The increases depended significantly on the amount of transfected RNA. Small amounts of RNA or templates that replicated inefficiently showed more enhancement. The infectivity of infectious RNA was increased by at least 10-fold in cells expressing CP. Moreover, virus infectivity was greatly enhanced in such cells. In other cells that expressed higher levels of CP, RNA replication of replicons was inhibited. Thus, depending on conditions, CP can markedly enhance or inhibit RUB RNA replication.     

Defective Interfering Influenza Virus RNAs: Time To Reevaluate Their Clinical Potential as Broad-Spectrum Antivirals?

Defective interfering (DI) RNAs are highly deleted forms of the infectious genome that are made by most families of RNA viruses. DI RNAs retain replication and packaging signals, are synthesized preferentially over infectious genomes, and are packaged as DI virus particles which can be transmitted to susceptible cells. Their ability to interfere with the replication of infectious virus in cell culture and their potential as antivirals in the clinic have long been known. However, until now, no realistic formulation has been described. In this review, we consider the early evidence of antiviral activity by DI viruses and, using the example of DI influenza A virus, outline developments that have led to the production of a cloned DI RNA that is highly active in preclinical studies not only against different subtypes of influenza A virus but also against heterologous respiratory viruses. These data suggest the timeliness of reassessing the potential of DI viruses as a novel class of antivirals that may have general applicability.     

Homologous interference of lymphocytic choriomeningitis virus: detection and measurement of interference focus-forming units.

Lymphocytic choriomeninigitis (LCM) virus defective interfering (DI) particles form foci of protected cells in a monolayer under an agarose-containing overlay medium. Foci originate from one cell dually infected with at least 1 interference focus-forming unit and infectious virus. As a result, an interfering factor is produced and released which interacts with neighboring cells, thereby protecting them against cytopathic lysis by challenge virus. The property of individual LCM virus DI particles to induce countable foci has been made the basis of quantitative assay that is comparable in every respect to the plaque assay of infectious virus and is much more sensitive and probably more accurate than other procedures used to measure LCM virus DI particles. LCM virus was passaged, undiluted, 10 times in cell cultures. When yields were analyzed as to concentrations of PFU and interference focus-forming units, both entities were found to fluctuate with the pattern expected from theoretical considerations.     

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.     

Characterization of homologous defective interfering RNA during persistent infection of Vero cells with Japanese encephalitis virus

It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese en-cephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homolo-gous DI virus intimately associated with JEV persis-tence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a per-sistent JEV infection in which the DI RNA co-replicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated dur-ing its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identifi-cation of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules re-tained their open reading frames despite a large dele-tion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these ob-servations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection.     

Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus

The genomic viral RNA (vRNA) segments of influenza A virus contain specific packaging signals at their termini that overlap the coding regions. To further characterize cis-acting signals in segment 7, we introduced synonymous mutations into the terminal coding regions. Mutation of codons that are normally highly conserved reduced virus growth in embryonated eggs and MDCK cells between 10- and 1,000-fold compared to that of the wild-type virus, whereas similar alterations to nonconserved codons had little effect. In all cases, the growth-impaired viruses showed defects in virion assembly and genome packaging. In eggs, nearly normal numbers of virus particles that in aggregate contained apparently equimolar quantities of the eight segments were formed, but with about fourfold less overall vRNA content than wild-type virions, suggesting that, on average, fewer than eight segments per particle were packaged. Concomitantly, the particle/PFU and segment/PFU ratios of the mutant viruses showed relative increases of up to 300-fold, with the behavior of the most defective viruses approaching that predicted for random segment packaging. Fluorescent staining of infected cells for the nucleoprotein and specific vRNAs confirmed that most mutant virus particles did not contain a full genome complement. The specific infectivity of the mutant viruses produced by MDCK cells was also reduced, but in this system, the mutations also dramatically reduced virion production. Overall, we conclude that segment 7 plays a key role in the influenza A virus genome packaging process, since mutation of as few as 4 nucleotides can dramatically inhibit infectious virus production through disruption of vRNA packaging.     

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.     

A second functional RNA domain in the 5' UTR of the Tomato bushy stunt virus genome: intra- and interdomain interactions mediate viral RNA replication

The 5' untranslated regions (UTRs) of (+)-strand RNA viruses play a variety of roles in the reproductive cycles of these infectious agents. Tomato bushy stunt virus (TBSV) belongs to this class of RNA virus and is the prototype member of the genus Tombusvirus. Previous studies have demonstrated that a T-shaped domain (TSD) forms in the 5' half of the TBSV 5' UTR and that it plays a central role in viral RNA replication. Here we have extended our structure-function analysis to the 3' half of the 5' UTR. Investigation of this region in the context of a model viral replicon (i.e., a TBSV-derived defective interfering [DI] RNA) revealed that this segment contains numerous functionally relevant structural features. In vitro solution structure probing along with comparative and computer-aided RNA secondary structure analyses predicted the presence of a simple stem loop (SL5) followed by a more complex downstream domain (DSD). Both structures were found to be essential for efficient DI RNA accumulation when tested in a plant protoplast system. For SL5, maintenance of the base of its stem was the principal feature required for robust in vivo accumulation. In the DSD, both helical and unpaired regions containing conserved sequences were necessary for efficient DI RNA accumulation. Additionally, optimal DI RNA accumulation required a TSD-DSD interaction mediated by a pseudoknot. Modifications that reduced accumulation did not appreciably affect DI RNA stability in vivo, indicating that the DSD and SL5 act to facilitate viral RNA replication.     

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.     

Bovine viral diarrhea virus: characterization of a cytopathogenic defective interfering particle with two internal deletions.

Molecular characterization of bovine viral diarrhea virus pair 13 revealed that isolate CP13 is composed of a cytopathogenic (cp) defective interfering particle (DI13) and a noncytopathogenic (noncp) helper virus. The DI13 genome possesses two internal deletions of 1,611 and 3,102 nucleotides. Except for a small fragment of the gene coding for glycoprotein E1, all structural protein genes are deleted together with most of the Npro gene, the region coding for nonstructural proteins p7 and NS2. While the amino terminus of NS3 seems to be strictly conserved for all other cp bovine viral diarrhea viruses, NS3 of DI13 is amino-terminally truncated and fused to 23 amino acids derived from Npro and E1. Characterization of the DI-helper virus system revealed a striking discrepancy between RNA production and generation of infectious viruses.     

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.