Recombination and Coronavirus Defective Interfering RNAs

Naturally occurring defective interfering RNAs have been found in 4 of 14 coronavirus species. They range in size from 2.2 kb to approximately 25 kb, or 80% of the 30-kb parent virus genome. The large DI RNAs do not in all cases appear to require helper virus for intracellular replication and it has been postulated that they may on their own function as agents of disease. Coronavirus DI RNAs appear to arise by internal deletions (through nonhomologous recombination events) on the virus genome or on DI RNAs of larger size by a polymerase strand-switching (copy-choice) mechanism. In addition to their use in the study of virus RNA replication and virus assembly, coronavirus DI RNAs are being used in a major way to study the mechanism of a high-frequency, site-specific RNA recombination event that leads to leader acquisition during virus replication (i.e., the leader fusion event that occurs during synthesis of subgenomic mRNAs, and the leader-switching event that can occur during DI RNA replication), a distinguishing feature of coronaviruses (and arteriviruses). Coronavirus DI RNAs are also being engineered as vehicles for the generation of targeted recombinants of the parent virus genome.     

Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging

Defective-interfering (DI) genomes of a virus contain sequence information essential for their replication and packaging. They need not contain any coding information and therefore are a valuable tool for identifying cis-acting, regulatory sequences in a viral genome. To identify these sequences in a DI genome of Sindbis virus, we cloned a cDNA copy of a complete DI genome directly downstream of the promoter for the SP6 bacteriophage DNA dependent RNA polymerase. The cDNA was transcribed into RNA, which was transfected into chicken embryo fibroblasts in the presence of helper Sindbis virus. After one to two passages the DI RNA became the major viral RNA species in infected cells. Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide region at the 3' terminus are specifically required for replication and packaging of these genomes.     

Deletion mapping of a mouse hepatitis virus defective interfering RNA reveals the requirement of an internal and discontiguous sequence for replication.

All of the defective interfering (DI) RNAs of mouse hepatitis virus (MHV) contain both the 5' and 3' ends of the viral genomic RNA, which presumably include the cis sequences required for RNA replication. To define the replication signal of MHV RNA, we have used a vaccinia virus-T7 polymerase-transcribed MHV DI RNA to study the effects of sequence deletion on DI RNA replication. Following infection of susceptible cells with a recombinant vaccinia virus expressing T7 RNA polymerase, various cDNA clones derived from a DI RNA (DIssF) of the JHM strain of MHV, which is a 3.5-kb naturally occurring DI RNA, behind a T7 promoter were transfected. On superinfection with a helper MHV, the ability of various DI RNAs to replicate was determined. Serial deletions from the middle of the RNA toward both the 5' and 3' ends demonstrated that 859 nucleotides from the 5' end and 436 nucleotides from the 3' end of the MHV RNA genome were necessary for RNA replication. Surprisingly, an additional stretch of 135 nucleotides located at 3.1 to 3.3 kb from the 5' end of the genome was also required. This stretch is discontiguous from the 5'-end cis replication signal and is present in all of the naturally occurring DI RNAs studied so far. The requirement for a long stretch of 5'- and 3'-end sequences predicts that the subgenomic MHV mRNAs cannot replicate. The efficiency of RNA replication varied with different cDNA constructs, suggesting possible interaction between different regions of DI RNA. The identification of MHV RNA replication signals allowed the construction of an MHV DI-based expression vector, which can express foreign genes, such as the chloramphenicol acetyltransferase gene.     

Cell culture-based production of defective interfering particles for influenza antiviral therapy

Defective interfering particles (DIPs) lack an essential portion of the virus genome, but retain signals for replication and packaging, and therefore, interfere with standard virus (STV) replication. Due to this property, DIPs can be potential antivirals. The influenza A virus DIP DI244, generated during propagation in chicken eggs, has been previously described as a potential candidate for influenza antiviral therapy. As a cell culture-based manufacturing process would be more suitable to fulfill large-scale production needs of an antiviral and enables full process control in closed systems, we investigated options to produce DI244 in the avian cell line AGE1.CR.pIX in chemically defined suspension culture. With a DI244 fraction of 55.8% compared to STV, the highest DI244 yield obtained from 50 million cells was 4.6 × 10⁹ vRNA copies/mL at 12 h post infection. However, other defective genomes were also detected. Since these additionally produced defective particles are non-infectious, they might be still useful in antiviral therapies. In case they would interfere with quality of the final product, we examined the impact of virus seeds and selected process parameters on DI244 yield and contamination level with other defective particles. With a DI244 fraction of 5.5%, the yield obtained was 1.7 × 10⁸ vRNA copies/mL but now without additional defective genomes. Although the DI244 yield might be decreased in this case, such controlled manufacturing conditions are not available in chicken eggs. Overall, the application of these findings can support design and optimization of a cell culture-based production process for DIPs to be used as antivirals.

     

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.     

The fitness of defective interfering murine coronavirus DI-a and its derivatives is decreased by nonsense and frameshift mutations.

The genome of the defective interfering (DI) mouse hepatitis virus DI-a carries a large open reading frame (ORF) consisting of ORF1a, ORF1b, and nucleocapsid sequences. To test whether this fusion ORF is important for DI virus replication, we constructed derivatives of the DI-a genome in which the reading frame was truncated by a nonsense codon or a frameshift mutation. In vitro-transcribed DI RNAs were transfected into mouse hepatitis virus-infected cells followed by undiluted passage of the resulting virus-DI virus stocks. The following observations were made. (i) Truncation of the fusion ORF was not lethal but led to reduced accumulation of DI RNA. (ii) When pairs of nearly identical in-frame and out-of-frame DI RNAs were directly compared by cotransfection, DI viruses containing in-frame genomic RNAs prevailed within three successive passage even when the out-of-frame RNAs were transfected in 10-fold molar excess. (iii) When DI viruses containing out-of-frame genomic RNAs were passaged, mutants emerged and were selected for that had restored the reading frame. We conclude that translation of the fusion ORF is indeed required for efficient propagation of DI-a and its derivatives.     

Sequence analysis of cDNA''s derived from the RNA of Sindbis virions and of defective interfering particles

Sindbis virus generates defective interfering (DI) particles during serial high-multiplicity passage in cultured cells. These DI particles inhibit the replication of infectious virus and can be an important factor in the establishment and maintenance of persistent infection in BHK cells. In an effort to understand how these DI particles are generated and how they interfere with the replication of standard virus, we performed a partial sequence analysis of the RNA obtained from two independently isolated populations of DI particles and from two Sindbis virus variants and compared these with the RNA of the parental wild-type virus. The 3'-terminal regions of the RNAs were sequenced by the dideoxy chain terminating method. Internal regions of the RNA were examined by restriction endonuclease digestion of cDNA's made to the various RNAs and by direct chemical sequencing of 5' end-labeled restriction fragments from cDNA made to the DI RNAs. One of the variant viruses examined was originally derived from cells persistently infected with Sindbis virus for 16 months and is resistant to interference by the DI strains used. In the 3'-terminal region of the RNA from this variant, only two base changes were found; one of these occurs in the 20-nucleotide 3'-terminal sequence which is highly conserved among alphaviruses. The DI RNA sequences were found to have been produced not by a single deletional event, but by multiple deletion steps combined with sequence rearrangements; all sequences examined are derived from the plus strand of Sindbis virion RNA. Both DI RNAs had at least 50 nucleotides of wild-type sequence conserved at the 3' terminus; in addition, they both contained conserved and perhaps amplified sequences derived from the non-26S region of the genome which may be of importance in their replication and interference ability.     

Pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by an internal deletion.

Cytopathogenic bovine viral diarrhea virus (BVDV) arises by RNA recombination in animals persistently infected with noncytopathogenic BVDV. Such animals develop fatal mucosal disease. In this report, the genome of a cytopathogenic BVDV isolate, termed CP9, is characterized. CP9-infected cells contained not only viral genomic RNA of 12.3 kb but also a BVDV-specific RNA of 8 kb. cDNA cloning and sequencing revealed that the 8-kb RNA is a BVDV genome with an internal deletion of 4.3 kb. The 8-kb RNA represents the genome of a typical defective interfering particle (DI), since its replication was strictly dependent on the presence of a helper virus and strongly interfered with the replication of the helper. Cell culture experiments demonstrated that the CP9 virus stock contains two viruses, namely, a helper virus and DI9. While the helper virus alone was noncytopathogenic, the presence of the DI conferred cytopathogenicity. Expression experiments demonstrated that p80, the marker protein of cytopathogenic BVDV, is translated from the defective genome. The occurrence of this cytopathogenic DI is linked to a fatal disease in cattle.     

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.     

Primary structure of poliovirus defective-interfering particle genomes and possible generation mechanisms of the particles

The genomes of defective-interfering (DI) particles derived from the Sabin strain of type 1 poliovirus (PV1(Sab] were characterized by nuclease S1 mapping using complementary DNA (cDNA) copies of PV1(Sab) genome as probes. The results demonstrated variety in the size and location of the deletions, which were compatible with our previous prediction. The results further indicated that the locations of the deletions were limited within the internal genome region encoding viral capsid proteins and that the deletion sites were clustered in certain areas on the genome. Sequence analysis of a number of cloned cDNAs to the DI genomes revealed that every DI genome retained the correct reading frame for viral protein synthesis. These results strongly suggested that one or all of the viral non-structural proteins might be cis-acting at least at a certain stage in viral replication. A computer search for secondary structures with regard to the deletion sites provided a possible common structure from which, supported by sequences existing on the plus or minus RNA strand of PV1(Sab), deletion regions looped out from the remaining sequences. Replicase might, therefore, skip these transiently formed loop structures with certain frequencies, resulting in the generation of DI genomes. This model could also be considered as a model for genetic recombination in these RNA genomes. Possible "supporting sequences" were also found for every rearranged site on the RNAs of influenza virus and sindbis virus. Thus, we propose a new copy-choice model, designated the "supporting sequence-loop model", for the generation of rearrangements occurring on single-stranded RNA genomes.     

Efficient homologous RNA recombination and requirement for an open reading frame during replication of equine arteritis virus defective interfering RNAs

Equine arteritis virus (EAV), the prototype arterivirus, is an enveloped plus-strand RNA virus with a genome of approximately 13 kb. Based on similarities in genome organization and protein expression, the arteriviruses have recently been grouped together with the coronaviruses and toroviruses in the newly established order Nidovirales. Previously, we reported the construction of pEDI, a full-length cDNA copy of EAV DI-b, a natural defective interfering (DI) RNA of 5.6 kb (R. Molenkamp et al., J. Virol. 74:3156-3165, 2000). EDI RNA consists of three noncontiguous parts of the EAV genome fused in frame with respect to the replicase gene. As a result, EDI RNA contains a truncated replicase open reading frame (EDI-ORF) and encodes a truncated replicase polyprotein. Since some coronavirus DI RNAs require the presence of an ORF for their efficient propagation, we have analyzed the importance of the EDI-ORF in EDI RNA replication. The EDI-ORF was disrupted at different positions by the introduction of frameshift mutations. These were found either to block DI RNA replication completely or to be removed within one virus passage, probably due to homologous recombination with the helper virus genome. Using recombination assays based on EDI RNA and full-length EAV genomes containing specific mutations, the rates of homologous RNA recombination in the 3'- and 5'-proximal regions of the EAV genome were studied. Remarkably, the recombination frequency in the 5'-proximal region was found to be approximately 100-fold lower than that in the 3'-proximal part of the genome.     

An internally located RNA hairpin enhances replication of Tomato bushy stunt virus RNAs

Defective interfering (DI) RNAs of Tomato bushy stunt virus (TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotide-long sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3' terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientation-independent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5'-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5'-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.     

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.