Defective Interfering RNA Accumulation Research Articles

Multiple Activities Associated with the Capsid Protein of Satellite Panicum Mosaic Virus Are Controlled Separately by the N- and C-Terminal Regions

The 17-kDa capsid protein (CP) of satellite panicum mosaic virus (SPMV) contains a distinct N-terminal arginine-rich motif (N-ARM) which is required for SPMV virion assembly and the activity of SPMV CP to promote systemic accumulation of its cognate RNA. The present study indicates that SPMV CP also is involved in SPMV RNA accumulation in inoculated leaves and that this activity is also dependent on a functional N-ARM. In addition, deletions of a C-terminal region abolish virion assembly and impair SPMV RNA accumulation in both inoculated and systemic leaves. Unlike the N-ARM mutations, substantial deletions of the SPMV CP C-terminus do not affect SPMV RNA binding activity. Interestingly, SPMV CP also binds Panicum mosaic virus genomic RNA via N-ARM-mediated CP:RNA interactions. Mutations of the N-ARM and the C-terminal regions significantly reduce SPMV CP titers and result in symptom attenuation. In contrast, virions were not associated per se with symptom exacerbation or successful SPMV RNA accumulation. The results show the existence of a correlation between N- and C-termini-mediated contributions for CP accumulation, symptom induction, defective-interfering RNA accumulation, and temperature sensitivity of SPMV RNA maintenance. The data provide further evidence that SPMV CP has multiple roles during infection, which might involve the formation of nonvirion CP:RNA complexes whose stability is controlled in a biologically relevant manner by the N- and C-termini of the CP.

Effect of Mutations in the Mouse Hepatitis Virus 3′(+)42 Protein Binding Element on RNA Replication

The mouse hepatitis virus (MHV) genome's 3' untranslated region contains cis-acting sequences necessary for replication. Studies of MHV and other coronaviruses have indicated a role for RNA secondary and tertiary elements in replication. Previous work in our laboratory has identified four proteins which form ribonucleoprotein complexes with the 3'-terminal 42 nucleotides [3'(+)42] of the MHV genome. Defective interfering (DI) RNA replication assays have demonstrated a role for the 3'(+)42 host protein binding element in the MHV life cycle. Using gel mobility shift RNase T1 protection assays and secondary structure modeling, we have characterized a possible role for RNA secondary structure in host protein binding to the 3'-terminal 42-nucleotide element. Additionally we have identified a role for the 3'-terminal 42-nucleotide host protein binding element in RNA replication and transcription using DI RNA replication assays and targeted recombination and by directly constructing mutants in this protein binding element using a recently described MHV reverse genetic system. DI RNA replication assays demonstrated that mutations in the 3'(+)42 host protein binding element had a deleterious effect on the accumulation of DI RNA. When the identical mutations were directly inserted into the MHV genome, most mutant genomes were viable but formed smaller plaques than the wild-type parent virus. One mutant was not viable. This mutant directed the synthesis of genome-sized negative-sense RNA approximately as efficiently as the wild type did but had a defect in subgenomic mRNA synthesis. These results point to a potential role for sequences at the extreme 3' end of the MHV genome in subgenomic RNA synthesis.

Effect of the Host and Temperature on the Formation of Defective RNAs Associated with Broad bean mottle virus Infection

ABSTRACT Previously, we demonstrated that Broad bean mottle virus (BBMV), a member of the genus Bromovirus, could accumulate RNA 2-derived defective interfering (DI) RNAs during infection. In this work, we study how host and environmental factors affect the accumulation of DI RNAs. Serial passages of BBMV through selected plant species reveal that, with low-multiplicity inocula, some systemic hosts (Vicia faba, Nicotiana clevelandii, and N. tabacum cv. Samsum) support DI RNA accumulation after the first passage cycle but other hosts (Phaseolus vulgaris, Pisum sativum, and Glycine max) do not. However, several passages with the high-multiplicity inocula can generate DI RNAs in pea plants. Local lesion hosts (Chenopodium quinoa, C. amaranticolor, and C. murale) remain free of the DI RNA components. The size of the de novo-formed DI RNAs depends on the host and on environmental conditions. For instance, broad bean plants cultivated in a greenhouse or in a growth chamber at 20 degrees C accumulated DI RNAs of 2.4 or 1.9 kb in size, respectively. A reverse trend was observed in pea plants. Lower temperatures greatly facilitated the formation of DI RNAs in broad bean and pea hosts after the first passage. The importance of these findings for the studies on DI RNAs are discussed.

3′-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs

The plus-strand RNA genome of tomato bushy stunt virus (TBSV) contains a 351-nucleotide (nt)-long 3′-untranslated region. We investigated the role of the 3′-proximal 130 nt of this sequence in viral RNA accumulation within the context of a TBSV defective interfering (DI) RNA. Sequence comparisons between different tombusviruses revealed that the 3′ portion of the 130-nt sequence is highly conserved and deletion analysis confirmed that this segment is required for accumulation of DI RNAs in protoplasts. Computer-aided sequence analysis and in vitro solution structure probing indicated that the conserved sequence consists of three stem-loop (SL) structures (5′-SL3-SL2-SL1-3′). The existence of SLs 1 and 3 was also supported by comparative secondary structure analysis of sequenced tombusvirus genomes. Formation of the stem regions in all three SLs was found to be very important, and modification of the terminal loop sequences of SL1 and SL2, but not SL3, decreased DI RNA accumulation in vivo. For SL3, alterations to an internal loop resulted in significantly reduced DI RNA levels. Collectively, these data indicate that all three SLs are functionally relevant and contribute substantially to DI RNA accumulation. In addition, secondary structure analysis of other tombusvirus replicons and related virus genera revealed that a TBSV satellite RNA and members of the closely related genus Aureusvirus (family Tombusviridae) share fundamental elements of this general structural arrangement. Thus, this secondary structure model appears to extend beyond tombusvirus genomes. These conserved 3′-terminal RNA elements likely function in vivo by promoting and/or regulating minus-strand synthesis.

Stem-loop III in the 5' untranslated region is a cis-acting element in bovine coronavirus defective interfering RNA replication.

Higher-order structures in the 5' untranslated region (UTR) of plus-strand RNA viruses are known in many cases to function as cis-acting elements in RNA translation, replication, or transcription. Here we describe evidence supporting the structure and a cis-acting function in defective interfering (DI) RNA replication of stem-loop III, the third of four predicted higher-order structures mapping within the 210-nucleotide (nt) 5' UTR of the 32-kb bovine coronavirus (BCoV) genome. Stem-loop III maps at nt 97 through 116, has a calculated free energy of -9.1 kcal/mol in the positive strand and -3.0 kcal/mol in the negative strand, and has associated with it beginning at nt 100 an open reading frame (ORF) potentially encoding an 8-amino-acid peptide. Stem-loop III is presumed to function in the positive strand, but its strand of action has not been established. Stem-loop III (i) shows phylogenetic conservation among group 2 coronaviruses and appears to have a homolog in coronavirus groups 1 and 3, (ii) has in all coronaviruses for which sequence is known a closely associated short, AUG-initiated intra-5' UTR ORF, (iii) is supported by enzyme structure-probing evidence in BCoV RNA, (iv) must maintain stem integrity for DI RNA replication in BCoV DI RNA, and (v) shows a positive correlation between maintenance of the short ORF and maximal DI RNA accumulation in BCoV DI RNA. These results indicate that stem-loop III in the BCoV 5' UTR is a cis-acting element for DI RNA replication and that its associated intra-5' UTR ORF may function to enhance replication. It is postulated that these two elements function similarly in the virus 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.

Size-dependent cell-to-cell movement of defective interfering RNAs of Cymbidium ringspot virus.

Co-inoculation of Nicotiana benthamiana plants with in vitro transcripts of both genomic and short defective interfering (DI) RNAs of Cymbidium ringspot virus results in an accumulation of de novo generated DI RNA dimers. Time-course analysis of DI RNA accumulation in the inoculated leaves showed early accumulation of DI RNA dimers followed by increased levels of DI RNA monomers. In contrast, DI RNA dimers were barely detectable in systems where cell-to-cell movement does not take place (protoplasts) or is less important (monomeric DI RNA-expressing transgenic plants). Our results also demonstrated that the size of DI RNAs is important in the colonization of inoculated leaves, suggesting that DI RNA dimers are quickly selected for cell-to-cell movement if short DI RNA monomers are used for infection.

Enhanced accumulation of coronavirus defective interfering RNA from expressed negative-strand transcripts by coexpressed positive-strand RNA transcripts.

Expression of negative-strand murine coronavirus mouse hepatitis virus (MHV) defective interfering (DI) RNA transcripts in MHV-infected cells results in the accumulation of positive-strand DI RNAs (M. Joo et al., 1996, J. Virol. 70, 5769–5776). However, the expressed negative-strand DI RNA transcripts are poor templates for positive-strand DI RNA synthesis. The present study demonstrated that DI RNA accumulation from the expressed negative-strand DI RNA transcripts in MHV-infected cells was enhanced by the coexpression of complementary RNA transcripts that correspond to the 5′ region of positive-strand DI RNA. The positive-strand RNA transcripts corresponding to the 5′ end-most 0.7–2.0 kb DI RNA had a similar enhancement effect. The coexpressed positive-strand RNA transcripts lacking the leader sequence or those containing only the leader sequence failed to demonstrate this enhancement effect, demonstrating that the presence of the leader sequence in the coexpressed positive-strand RNA transcripts was necessary, but not sufficient, for the enhancement of DI RNA accumulation from the coexpressed negative-strand DI RNA transcripts. Negative-strand DI RNA transcripts that were coexpressed with the partial-length positive-strand RNA transcripts were no more stable than those expressed alone, suggesting that a higher stability of the expressed negative-strand RNA transcripts was an unlikely reason for the higher DI RNA accumulation in cells coexpressing two complementary DI RNA transcripts. Sequence analyses unexpectedly demonstrated that the leader sequence of the majority of accumulated DI RNAs switched to helper virus derived leader sequence, suggesting that enhancement of DI RNA accumulation was mediated by the efficient utilization of helper virus derived leader sequence for DI RNA synthesis. Furthermore, our data suggested that this leader switching, a type of homologous RNA–RNA recombination, occurred during positive-strand DI RNA synthesis and that MHV positive-strand RNA synthesis mechanism may have a preference toward recognizing double-stranded RNA structures over single-stranded negative-strand RNA to produce positive-strand DI RNAs.

An RNA domain within the 5′ untranslated region of the tomato bushy stunt virus genome modulates viral RNA replication

The terminal half of the 5′ untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in viral RNA replication. Computer-aided thermodynamic analysis of secondary structure, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5′-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a prominent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5′ or 3′ segment forming S1 (coordinates 1–10 or 69–78, respectively) in a model subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20-fold. Compensatory-type mutational analysis of S1 within this replicon revealed a strong correlation between formation of the predicted S1 structure and efficient DI RNA accumulation. RNA decay studies in vivodid not reveal any notable changes in the physical stabilities of DI RNAs containing disrupted S1s, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was significantly more detrimental to DI RNA accumulation than (−)-strand destabilization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5′-proximal secondary structure of a previously identified mutant DI RNA, RNA B, that lacks the 5′ UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5′-terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role for the TSD, and in particular its S1 subelement, in tombusvirus RNA replication.

Enhancer-like Properties of an RNA Element That Modulates Tombusvirus RNA Accumulation

Prototypical defective interfering (DI) RNAs of the plus-strand RNA virus tomato bushy stunt virus contain four noncontiguous segments (regions I–IV) derived from the viral genome. Region I corresponds to 5′-noncoding sequence, regions II and III are derived from internal positions, and region IV represents a 3′-terminal segment. We analyzed the internally located region III in a prototypical DI RNA to understand better its role in DI RNA accumulation. Our results indicate that (1) region III is not essential for DI RNA accumulation, but molecules that lack it accumulate at significantly reduced levels (∼10-fold lower), (2) region III is able to function at different positions and in opposite orientations, (3) a single copy of region III is favored over multiple copies, (4) the stimulatory effect observed on DI RNA accumulation is not due to region III-mediated RNA stabilization, (5) DI RNAs lacking region III permit the efficient accumulation of head-to-tail dimers and are less effective at suppressing helper RNA accumulation, and (6) negative-strand accumulation is also significantly depressed for DI RNAs lacking region III. Collectively, these results support a role for region III as an enhancer-like element that facilitates DI RNA replication. A scanning-type mutagenesis strategy was used to define portions of region III important for its stimulatory effect on DI RNA accumulation. Interestingly, the results revealed several differences in the requirements for activity when region III was in the forward versus the reverse orientation. In the context of the viral genome, region III was found to be essential for biological activity. This latter finding defines a critical role for this element in the reproductive cycle of the virus.

Characterization of the molecular mechanism of defective interfering RNA-mediated symptom attenuation in tombusvirus-infected plants.

Different tombusviruses were able to support the replication of either homologous or heterologous defective interfering (DI) RNAs, and those infected plants usually developed typical attenuated symptoms. However, in some helper virus-DI RNA combinations the inoculated plants were necrotized, although they contained a high level of DI RNA, suggesting that the accumulation of DI RNA and the resulting suppression of genomic RNA replication were not directly responsible for the symptom attenuation. Moreover, the 19-kDa protein product of ORF 5, which is known to play a crucial role in necrotic symptom development, accumulated at the same level in the infected plants in the presence of protective homologous DI RNA and in the presence of nonprotective heterologous DI RNA. It was also demonstrated, by chimeric helper viruses, that the ability of heterologous DI RNA to protect the virus-infected plants against systemic necrosis is determined by the 5'-proximal region of the helper virus genome. The results presented suggest that DI RNA-mediated protection did not operate via the specific inhibition of 19-kDa protein expression but, more likely, DI RNAs in protective DI-helper virus combinations specifically interacted with viral products, preventing the induction of necrotic symptoms.

Studies of murine coronavirus DI RNA replication from negative-strand transcripts.

The positive-strand transcripts as well as negative-strand transcripts of mouse hepatitis virus (MHV) defective interfering (DI) RNA, when introduced into MHV-infected cells, resulted in DI RNA replication and accumulation. The leader sequence of the majority of DI RNAs that accumulated from the expression of negative-strand DI RNA transcripts with no extra non-MHV nucleotides at the 3' end switched to that of helper virus, whereas this leader sequence switching did not occur in most of the positive-strand DI RNAs that accumulated from the expressed negative-strand DI RNA transcripts with extra non-MHV nucleotides at the 3' end. These data demonstrated that the extra 4 nucleotides at the 3'-end of negative-strand DI RNA transcripts affected leader sequence switching on DI RNA, and indicated that the leader switching probably occurred during positive-strand DI RNA synthesis.

Effect of 5′ and 3′ Terminal Sequences, Overall Length, and Coding Capacity on the Accumulation of Defective RNAs Associated with Broad Bean Mottle Bromovirusin Planta

Broad bean mottle bromovirus (BBMV) was shown to accumulate RNA2-derived defective interfering (DI) RNAs [Romeroet al., Virology194, 576–584 (1993); Poganyet al., Virology212, 574–586 (1995)]. In this work, we utilize three sets of BBMV RNA2-derived artificial DI RNA constructs to determine factors that affect the accumulation of defective RNAsin planta.One set of deletion constructs was used to localize sequences required for efficient accumulation within the 5′ 883 nt and the 3′ 387 nt of the DI RNAs. The second set had a gradually increasing size of 3′ nested deletions to determine the minimal length required for efficient DI RNA accumulation. The smallest DI RNA still accumulating in plants was found to be 1712 nt long. The third set consisted of frameshift mutants which showed that at least 64.4% of BBMV DI RNA sequences must have the 5′ portion of the 2a open reading frame to ensure efficient accumulation. The importance of these factors in the selection of DI RNAs is discussed.

Replication of murine coronavirus defective interfering RNA from negative-strand transcripts.

The positive-strand defective interfering (DI) RNA of the murine coronavirus mouse hepatitis virus (MHV), when introduced into MHV-infected cells, results in DI RNA replication and accumulation. We studied whether the introduction of negative-strand transcripts of MHV DI RNA would also result in replication. At a location downstream of the T7 promoter and upstream of the human hepatitis delta virus ribozyme domain, we inserted a complete cDNA clone of MHV DI RNA in reverse orientation; in vitro-synthesized RNA from this plasmid yielded a negative-strand RNA copy of the MHV DI RNA. When the negative-strand transcripts of the DI RNA were expressed in MHV-infected cells by a vaccinia virus T7 expression system, positive-strand DI RNAs accumulated in the plasmid-transfected cells. DI RNA replication depended on the expression of T7 polymerase and on the presence of the T7 promoter. Transfection of in vitro-synthesized negative-strand transcripts into MHV-infected cells and serial passage of virus samples from RNA-transfected cells also resulted in accumulation of the DI RNA. Positive-strand DI RNA transcripts were undetectable in sample preparations of the in vitro-synthesized negative-strand DI RNA transcripts, and DI RNA did not accumulate after cotransfection of a small amount of positive-strand DI RNA and truncated-replication-disabled negative-strand transcripts; clearly, the DI RNA replicated from the transfected negative-strand transcripts and not from minute amounts of positive-strand DI RNAs that might be envisioned as artifacts of T7 transcription. Sequence analysis of positive-strand DI RNAs in the cells transfected with negative-strand transcripts showed that DI RNAs maintained the DI-specific unique sequences introduced within the leader sequence. These data indicated that positive-strand DI RNA synthesis occurred from introduced negative-strand transcripts in the MHV-infected cells; this demonstration, using MHV, of DI RNA replication from transfected negative-strand DI RNA transcripts is the first such demonstration among all positive-stranded RNA viruses.

Tandem Placement of a Coronavirus Promoter Results in Enhanced mRNA Synthesis from the Downstream-Most Initiation Site

Insertion of the 17-nucleotide promoter region for the bovine coronavirus N gene as part of a 27-nucleotide cassette into the open reading frame of a cloned synthetic defective-interfering (DI) RNA resulted in synthesis of subDI RNA transcripts from the replicating DI RNA genome. Duplicating and triplicating the promoter sequence in tandem caused a progressive increase in the efficiency of subgenomic mRNA synthesis despite a concurrent decrease in the rate of DI RNA accumulation that was not specific to the promoter sequences being added. Although initiation of transcription (leader fusion) occurred at each of the three promoter sites in the tandem construct, almost all of the transcripts were found as a product of the most downstream (3′-most on the genome) promoter. These results show that enhancement of subgenomic mRNA synthesis is a property that can reside within sequence situated near the promoter. A possible role for the plus strand in the downstream promoter choice is suggested.

Cis Requirement for N-specific protein sequence in bovine coronavirus defective interfering RNA replication.

A naturally occurring 2.2-kb defective interfering (DI) RNA of the bovine coronavirus, structurally a simple fusion of the genomic termini, contains a single contiguous open reading frame (ORF) or 1.7 kb composed of the 5'-terminal 288 nucleotides of polymerase gene 1a and all 1,344 nucleotides of the nucleocapsid protein (N) gene. The ORF must remain open throughout most of its sequence for replication to occur. To determine the qualitative importance of the N portion of the chimeric ORF in DI RNA replication, transcripts of mutated reporter-containing constructs were tested for replication in helper virus-infected cells. It was determined that the N ORF could not be replaced by the naturally occurring internal I protein ORF, accomplished by deleting the first base in the N start codon which leads to a +1 frameshift, nor could it be replaced by the chloramphenicol acetyltransferase ORF. Furthermore, 3'-terminal truncations of the N gene leaving less than 85% of its total length were likewise not tolerated. Small in-frame deletions and in-frame foreign sequence insertions of up to 99 nucleotides within certain regions of the N ORF were tolerated, however, but the rate of DI RNA accumulation in these cases was lower. These results indicate that there is a requirement for translation of most if not all of the N protein in cis for optimal replication of the bovine coronavirus DI RNA and suggest that a similar requirement may exist for viral genome replication.

3′ Terminal Putative Stem–Loop Structure Required for the Accumulation of Cymbidium Ringspot Viral RNA

Defective interfering (DI) RNA of cymbidium ringspot tombusvirus (CymRSV) was used to identify thecis-acting nature of the last 77-nt sequence of the viral genome which is required for DI RNA accumulation. The 3′-terminalcis-essential domain of both genomic and DI RNAs can be folded into a stable stem–loop structure composed of three hairpins and two short non-base-paired regions. None of the three conserved stem–loops can be deleted without abolishing the infectivity of DI RNA. Similarly, those mutants in which base-paired stem regions were disrupted by single-, double-, or triple-base substitutions were unable to replicate. However, when the original structures were reconstructed by compensatory mutations the viability of the molecules was also restored. Limited mutation (1 or 2 nt) in the non-base-paired region did not show any significant effect on viral replication. Our results strongly suggest that the proposed structure for the 3′ terminus of the viral genome is very important for viral RNA replication. It is very likely that the function of this structure is to promote the minus-strand synthesis of CymRSV DI RNA. Evidence is provided that the proposed 3′-terminal structure is relevant not only for CymRSV DI but for genomic RNA as well.

Coat protein of cucumber necrosis virus is not required for efficient generation or accumulation of defective interfering RNAs

It is generally assumed that defective interfering (DI) forms of viruses are encapsidated in structural proteins encoded by the helper virus. Virion RNA extracts from cucumber necrosis virus (CNV) infections showing high levels of cellular DI RNAs contain barely detectable levels of DI RNAs, suggesting that DI RNAs are encapsidated very inefficiently. In addition, accumulation of CNV DI RNAs occurs at equal efficiency in coinoculated plants using either synthetic wild-type CNV genomic RNA as helper or a mutant of CNV which lacks the coat protein-coding sequence. Together this shows that the CNV coat protein is not required for efficient accumulation of CNV DI RNA in plants. Factors that could account for the high level of CNV DI RNAs in plants are discussed.

Nonhomologous RNA recombination in tombusviruses: generation and evolution of defective interfering RNAs by stepwise deletions

We used a protoplast system to study the mechanisms involved in the generation and evolution of defective interfering (DI) RNAs of tomato bushy stunt tombusvirus (TBSV). Synthetic transcripts corresponding to different naturally occurring TBSV DI RNAs, or to various artificially constructed TBSV defective RNAs, were analyzed. The relative levels of competitiveness of different DI RNAs were determined by coinoculating their corresponding transcripts into protoplasts along with helper genomic RNA transcripts and monitoring the level of DI RNA accumulation. Further studies were performed to assess the contribution of naked DI RNA stability and DI RNA encapsidation efficiency to the observed levels of competitiveness. In addition, the ability of various defective RNAs to evolve to alternative forms was tested by serially passaging protoplast infections initiated with transcripts corresponding to helper genomic RNA and a single type of defective RNA. These studies, and the analysis of the sequences of observed recombinants, indicate that (i) replication competence is a major factor dictating DI RNA competitiveness and is likely a primary determinant in DI RNA evolution, (ii) DI RNAs are capable of evolving to both smaller and larger forms, and the rates at which various transitions occur differ, (iii) DI RNA-DI RNA recombination and/or rearrangement is responsible for the formation of the evolved RNA molecules which were examined, and (iv) sequence complementarities between positive- and negative-sense strands in the regions of the junctions suggest that, in some cases, base pairing between an incomplete replicase-associated nascent strand and acceptor template may mediate selection of recombination sites. On the basis of our data, we propose a stepwise deletion model to describe the temporal order of events leading to the formation of tombusvirus DI RNAs.

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.