Conserved regions in defective interfering viral double-stranded RNAs from a yeast virus.

We have completely sequenced a defective interfering viral double-stranded RNA (dsRNA) from the Saccharomyces cerevisiae virus. This RNA (S14) is a simple internal deletion of its parental dsRNA, M1, of 1.9 kilobases. The 5' 964 bases of the M1 plus strand encode the type 1 killer toxin of the yeast. S14 is 793 base pairs (bp) long, with 253 bp from the 5' region of its parental plus strand and 540 bp from the 3' region. All three defective interfering RNAs derived from M1 that have been characterized so far preserve a large 3' region, which includes five repeats of a rotationally symmetrical 11-bp consensus sequence. This 11-bp sequence is not present in the 5' 1 kilobase of the parental RNA or in any of the sequenced regions of unrelated yeast viral dsRNAs, but it is present in the 3' region of the plus strand of another yeast viral dsRNA, M2, that encodes the type 2 killer toxin. The 3' region of 550 bases of the M1 plus strand, previously only partially sequenced, reveals no large open reading frames. Hence only about half of M1 appears to have a coding function.     

Translation but not the encoded sequence is essential for the efficient propagation of the defective interfering RNAs of the coronavirus mouse hepatitis virus.

The defective interfering (DI) RNA MIDI of mouse hepatitis virus strain A59 (MHV-A59) contains a large open reading frame (ORF) spanning almost its entire genome. This ORF consists of sequences derived from ORF1a, ORF1b, and the nucleocapsid gene. We have previously demonstrated that mutations that disrupt the ORF decrease the fitness of MIDI and its derivatives (R. J. de Groot, R. G. van der Most, and W. J. M. Spaan, J. Virol. 66:5898-5905, 1992). To determine whether translation of the ORF per se is required or whether the encoded polypeptide or a specific sequence is involved, we analyzed sets of related DI RNAs containing different ORFs. After partial deletion of ORF1b and nucleocapsid gene sequences, disruption of the remaining ORF is still lethal; translation of the entire ORF is not essential, however. When a large fragment of the MHV-A59 spike gene, which is not present in any of the MHV-A59 DI RNAs identified so far, was inserted in-frame into a MIDI derivative, translation across this sequence was vital to DI RNA survival. Thus, the translated sequence is irrelevant, indicating that translation per se plays a crucial role in DI virus propagation. Next, it was examined during which step of the viral life cycle translation plays its role. Since the requirement for translation also exists in DI RNA-transfected and MHV-infected cells, it follows that either the synthesis or degradation of DI RNAs is affected by translation.     

Comparative analysis of intracellular inhibition of hepatitis C virus replication by small interfering RNAs

Several synthetic siRNAs were designed to target various regions of hepatitis C virus (HCV) replicon RNA. The antiviral efficacies of the siRNAs were compared using real time PCR and western blot assessment. siRNAs targeting either specific coding region of HCV NS3 or NS5B were the most efficacious in terms of gene silencing and inhibitory activity of the HCV replicon replication. There was no activation of genes involved in innate immune response by the HCV-specific siRNA, indicating that HCV replication inhibition was not due to non-specific antiviral response. Moreover, 5′-RACE PCR analysis showed that the silencing effect by the siRNAs was mainly caused by specific cleavage of targeted HCV genomic RNA. These findings suggest that RNAi targeting HCV coding regions could provide a useful approach to anti-HCV treatment.     

Inhibition of intracellular hepatitis C virus replication by synthetic and vector-derived small interfering RNAs

Small interfering RNAs (siRNAs) efficiently inhibit gene expression by RNA interference. Here, we report efficient inhibition, by both synthetic and vector-derived siRNAs, of hepatitis C virus (HCV) replication, as well as viral protein synthesis, using an HCV replicon system. The siRNAs were designed to target the 5′ untranslated region (5′ UTR) of the HCV genome, which has an internal ribosomal entry site for the translation of the entire viral polyprotein. Moreover, the 5′ UTR is the most conserved region in the HCV genome, making it an ideal target for siRNAs. Importantly, we have identified an effective site in the 5′ UTR at which ~80% suppression of HCV replication was achieved with concentrations of siRNA as low as 2.5 nM. Furthermore, DNA-based vectors expressing siRNA against HCV were also effective, which might allow the efficient delivery of RNAi into hepatocytes in vivo using viral vectors. Our results support the feasibility of using siRNA-based gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.     

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.     

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.     

Guanidine-resistant defective interfering particles of poliovirus.

A mixture containing standard poliovirus and D3 particles (mutants with deletions in the capsid locus) was serially passaged in the presence of guanidine. Within five growth cycles, the standard virus was guanidine resistant, but the D3 particles were guanidine sensitive, even after 21 passages with the inhibitor. By passage 40 with guanidine, D3 particles were eliminated, and a new deletion mutant (DX) appeared in the virus population. D3 particles contained a 15% deletion, and DX particles contained a 6% deletion in the capsid locus. Although neither mutant induced the synthesis of NCVP1a or a complete complement of capsid proteins after infection, cells infected with DX particles produced two novel proteins, which had molecular weights of approximately 68,000 and 25,000.     

Induction and biological properties of defective interfering particles of rabies virus.

A method for obtaining large quantities of defective interfering (DI) rabies virus particles that fulfill all the criteria delineated by Huang and Baltimore (1970) is described. The purified rabies DI virion was found to be much shorter (60 to 80 nm) than the complete virion (180 nm) and to have a viral genome of about half the size of normal rabies RNA but with all of the structural proteins of standard virions. Rabies DI virions were noninfectious for both cells in culture and for animals. As determined by in vitro and in vivo techniques, interference with the replication of standard virus was specific to rabies virus. The possible role of rabies DI virion in the pathogenicity of rabies virus infection and in the establishment of attenuated strains for use as live rabies vaccines is discussed.     

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.     

RNA determinants of junction site selection in RNA virus recombinants and defective interfering RNAs.

RNA recombination plays an important role in the diversification and evolution of RNA viruses. Most of these events are believed to be mediated by an actively copying viral replicase switching from a donor template to an acceptor template, where it resumes synthesis. In addition, intramolecular replicase-mediated events (i.e., rearrangements) can lead to the generation of replicable deleted forms of a viral genome, termed defective interfering (DI) RNAs. To gain further insight into the recombination process, the effect of various primary and secondary structures on recombination site selection in vivo was examined using plant RNA tombusviruses. The effect of sequence identity and complementarity on deletion events that generate DI RNAs was also investigated. Our results suggest that (1) 5' termini and strong hairpin structures in donor templates represent preferred sites for recombinations, (2) junction sites in acceptor templates do not occur in double-stranded regions, (3) nucleotide homology can shift donor and acceptor recombination sites closer to regions of identity and, (4) both sequence identity and complementarity can direct deletion sites in DI RNAs. These results further define RNA determinants of tombusvirus RNA recombination and rearrangement.     

Silencing of hepatitis A virus infection by small interfering RNAs

Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequence-specific RNAi.     

Interference of hepatitis A virus replication by small interfering RNAs

The rate of acute liver failure due to hepatitis A virus (HAV) has not decreased, and therapy of severe infections is still of major interest. Using a DNA-based HAV replicon cell culture system, we demonstrate that small interfering RNAs (siRNAs) targeted against viral sequences or a reporter gene contained in the viral genome specifically inhibit HAV RNA replication in HuhT7 cells. Combinations of siRNAs were more effective suppressors of HAV RNA replication. Also, siRNAs targeted against HAV 2C and 3D inhibited the expression of the respective protein. Expressions of endogenous beta-actin and double-stranded-specific RNA-activated serin/threonine kinase (PKR) were unaltered, demonstrating that the siRNA inhibitory effect was not connected to interferon inhibition, but rather was specifically targeted against HAV RNA. These results suggest that RNA interference might ultimately be useful in treatment of severe HAV infection with or without chronic liver diseases.     

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.     

Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence

Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5′ untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence.These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine.