The AU-rich RNA recombination hot spot sequence of Brome mosaic virus is functional in tombusviruses: implications for the mechanism of RNA recombination

RNA recombination can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. In this paper, we demonstrate that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. However, unlike BMV or retroviruses, where recombination usually occurred with precision between duplicated AU-rich sequences, the majority of TBSV DI RNA recombinants were imprecise. In addition, only one copy of the AU-rich sequence was essential to promote recombination in the DI RNA. The selection of junction sites was also influenced by a putative cis-acting element present in the DI RNA. We found that this RNA sequence bound to the TBSV replicase proteins more efficiently than did control nonviral sequences, suggesting that it might be involved in replicase "landing" during the template switching events. In summary, evidence is presented that a tombusvirus can use the recombination signal of BMV. This supports the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses.     

Mechanism of RNA recombination in carmo- and tombusviruses: evidence for template switching by the RNA-dependent RNA polymerase in vitro

RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligation-based RNA recombination was not detected in vitro. Demonstration of the ability of carmo- and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses.     

RNA recombination in brome mosaic virus, a model plus strand RNA virus.

Studies on the molecular mechanism of genetic recombination in RNA viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. The system of brome mosaic virus (BMV) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of RNA-RNA recombination events. By using engineered BMV RNA components, the occurrence of both homologous and nonhomologous crosses were demonstrated among the segments of the BMV RNA genome. Studies show that the two types of crossovers require different RNA signal sequences and that both types depend upon the participation of BMV replicase proteins. Mutations in the two BMV-encoded replicase polypeptides (proteins 1a and 2a) reveal that their different regions participate in homologous and in nonhomologous crossovers. Based on all these data, it is most likely that homologous and nonhomologous recombinant crosses do occur via two different types of template switching events (copy-choice mechanism) where viral replicase complex changes RNA templates during viral RNA replication at distinct signal sequences. In this review we discuss various aspects of the mechanism of RNA recombination in BMV and we emphasize future projections of this research.     

Homologous RNA recombination in brome mosaic virus: AU-rich sequences decrease the accuracy of crossovers.

Brome mosaic virus, a tripartite positive-stranded RNA virus of plants, was used for the determination of sequence requirements of imprecise (aberrant) homologous recombination. A 23-nucleotide (nt) region that included a 6-nt UUAAAA sequence (designated the AU sequence) common between wild-type RNA2 and mutant RNA3 supported both precise and imprecise homologous recombination, though the latter occurred with lower frequency. Doubling the length of the 6-nt AU sequence in RNA3 increased the incidence of imprecise crossovers by nearly threefold. Duplication or triplication of the length of the AU sequence in both RNA2 and RNA3 further raised the frequency of imprecise crossovers. The majority of imprecise crosses were located within or close to the extended AU sequence. Imprecise recombinants contained either nucleotide substitutions, nontemplated nucleotides, small deletions, or small sequence duplications within the region of crossovers. Deletion of the AU sequence from the homologous region in RNA3 resulted in the accumulation of only precise homologous recombinants. Our results provide experimental evidence that AU sequences can facilitate the formation of imprecise homologous recombinants. The generation of small additions or deletions can be explained by a misannealing mechanism within the AU sequences, while replicase errors during RNA copying might explain the occurrence of nucleotide substitutions or nontemplated nucleotides.     

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.     

Role of RNA structure in non-homologous recombination between genomic molecules of brome mosaic virus

Brome mosaic virus (BMV) is a tripartite genome, positive-sense RNA virus of plants. Previously it was demonstrated that local hybridization between BMV RNAs (RNA–RNA heteroduplex formation) efficiently promotes non-homologous RNA recombination. In addition, studies on the role of the BMV polymerase in RNA recombination suggested that the location of non-homologous crossovers depends mostly on RNA structure. As a result, a detailed analysis of a large number of non-homologous recombinants generated in the BMV-based system was undertaken. Recombination hot-spots as well as putative elements in RNA structure enhancing non-homologous crossovers and targeting them in a site-specific manner were identified. To verify these observations the recombinationally active sequence in BMV RNA3 derivative was modified. The results obtained with new RNA3 mutants suggest that the primary and secondary structure of the sequences involved in a heteroduplex formation rather than the length of heteroduplex plays the most important role in the recombination process. The presented data indicate that the sequences proximal to the heteroduplex may also affect template switching by BMV replicase. Moreover, it was shown that both short homologous sequences and a hairpin structure have to accompany a double-stranded region to target non-homologous crossovers in a site-specific manner.     

Poliovirus RNA recombination: mechanistic studies in the absence of selection.

Direct and quantitative detection of recombinant RNA molecules by polymerase chain reaction (PCR) provides a novel method for studying recombination in RNA viruses without selection for viable progeny. The parental poliovirus strains used in this study contained polymorphic marker loci approximately 600 bases apart; both exhibited wild-type growth characteristics. We established conditions under which the amount of PCR product was linearly proportional to the amount of input template, and the reproducibility was high. Recombinant progeny were predominantly homologous and arose at frequencies up to 2 x 10(-3). Recombination events increased in frequency throughout replication, indicating that there is no viral RNA sequestration or inhibition of recombination late in infection as proposed in earlier genetic studies. Previous studies have demonstrated that poliovirus recombination occurs by a copy-choice mechanism in which the viral polymerase switches templates during negative-strand synthesis. Varying the relative amount of input parental virus markedly altered reciprocal recombination frequencies. This, in conjunction with the kinetics data, indicated that acceptor template concentration is a determinant of template switching frequency. Since positive strands greatly outnumber negative strands throughout poliovirus infection, this would explain the bias toward recombination during negative-strand synthesis.     

Emergence of distinct brome mosaic virus recombinants is determined by the polarity of the inoculum RNA

Despite overwhelming interest in the impact exerted by recombination during evolution of RNA viruses, the relative contribution of the polarity of inoculum templates remains poorly understood. Here, by agroinfiltrating Nicotiana benthamiana leaves, we show that brome mosaic virus (BMV) replicase is competent to initiate positive-strand [(+)-strand] synthesis on an ectopically expressed RNA3 negative strand [(-) strand] and faithfully complete the replication cycle. Consequently, we sought to examine the role of RNA polarity in BMV recombination by expressing a series of replication-defective mutants of BMV RNA3 in (+) or (-) polarity. Temporal analysis of progeny sequences revealed that the genetic makeup of the primary recombinant pool is determined by the polarity of the inoculum template. When the polarity of the inoculum template was (+), the recombinant pool that accumulated during early phases of replication was a mixture of nonhomologous recombinants. These are longer than the inoculum template length, and a nascent 3' untranslated region (UTR) of wild-type (WT) RNA1 or RNA2 was added to the input mutant RNA3 3' UTR due to end-to-end template switching by BMV replicase during (-)-strand synthesis. In contrast, when the polarity of the inoculum was (-), the progeny contained a pool of native-length homologous recombinants generated by template switching of BMV replicase with a nascent UTR from WT RNA1 or RNA2 during (+)-strand synthesis. Repair of a point mutation caused by polymerase error occurred only when the polarity of the inoculum template was (+). These results contribute to the explanation of the functional role of RNA polarity in recombination mediated by copy choice mechanisms.     

RNA recombination in a coronavirus: recombination between viral genomic RNA and transfected RNA fragments.

Mouse hepatitis virus (MHV), a coronavirus, has been shown to undergo a high frequency of RNA recombination both in tissue culture and in animal infection. So far, RNA recombination has been demonstrated only between genomic RNAs of two coinfecting viruses. To understand the mechanism of RNA recombination and to further explore the potential of RNA recombination, we studied whether recombination could occur between a replicating MHV RNA and transfected RNA fragments. We first used RNA fragments which represented the 5' end of genomic-sense sequences of MHV RNA for transfection. By using polymerase chain reaction amplification with two specific primers, we were able to detect recombinant RNAs which incorporated the transfected fragment into the 5' end of the viral RNA in the infected cells. Surprisingly, even the anti-genomic-sense RNA fragments complementary to the 5' end of MHV genomic RNA could also recombine with the MHV genomic RNAs. This observation suggests that RNA recombination can occur during both positive- and negative-strand RNA synthesis. Furthermore, the recombinant RNAs could be detected in the virion released from the infected cells even after several passages of virus in tissue culture cells, indicating that these recombinant RNAs represented functional virion RNAs. The crossover sites of these recombinants were detected throughout the transfected RNA fragments. However, when an RNA fragment with a nine-nucleotide (CUUUAUAAA) deletion immediately downstream of a pentanucleotide (UCUAA) repeat sequence in the leader RNA was transfected into MHV-infected cells, most of the recombinants between this RNA and the MHV genome contained crossover sites near this pentanucleotide repeat sequence. In contrast, when exogenous RNAs with the intact nine-nucleotide sequence were used in similar experiments, the crossover sites of recombinants in viral genomic RNA could be detected at more-downstream sites. This study demonstrated that recombination can occur between replicating MHV RNAs and RNA fragments which do not replicate, suggesting the potential of RNA recombination for genetic engineering.     

RNA-RNA recombination in Sindbis virus: roles of the 3' conserved motif, poly(A) tail, and nonviral sequences of template RNAs in polymerase recognition and template switching.

Sindbis virus (SIN), a mosquito-transmitted animal RNA virus, carries a 11.7-kb positive-sense RNA genome which is capped and polyadenylated. We recently reported that the SIN RNA-dependent RNA polymerase (RdRp) could initiate negative-strand RNA synthesis from a 0.3-kb 3'-coterminal SIN RNA fragment and undergo template switching in vivo (M. Hajjou, K. R. Hill, S. V. Subramaniam, J. Y. Hu, and R. Raju, J. Virol. 70:5153-5164, 1996). To identify and characterize the viral and nonviral sequences which regulate SIN RNA synthesis and recombination, a series of SIN RNAs carrying altered 3' ends were tested for the ability to produce infectious virus or to support recombination in BHK cells. The major findings of this report are as follows: (i) the 3'-terminal 20-nucleotides (nt) sequence along with the abutting poly(A) tail of the SIN genome fully supports negative-strand synthesis, genome replication, and template switching; (ii) a full-length SIN RNA carrying the 3'-terminal 24 nt but lacking the poly(A) tail is noninfectious; (iii) SIN RNAs which carry 3' 64 nt or more without the poly(A) tail are infectious and regain their poly(A) tail in vivo; (iv) donor templates lacking the poly(A) tail do not support template switching; (v) full-length SIN RNAs lacking the poly(A) tail but carrying 3' nonviral extensions, although debilitated to begin with, evolve into rapidly growing poly(A)-carrying mutants; (vi) poly(A) or poly(U) motifs positioned internally within the acceptor templates, in the absence of other promoter elements within the vicinity, do not induce the jumping polymerase to reinitiate at these sites; and (vii) the junction site selection on donor templates occurs independently of the sequences around the acceptor sites. In addition to furthering our understanding of RNA recombination, these studies give interesting clues as to how the alphavirus polymerase interacts with its 3' promoter elements of genomic RNA and nonreplicative RNAs. This is the first report that an in vitro-synthesized alphavirus RNA lacking a poly(A) tail can initiate infection and produce 3' polyadenylated viral genome in vivo.     

Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA

In our attempt to obtain further information on the replication mechanism of the hepatitis C virus (HCV), we have studied the role of sequences at the 3'-end of HCV minus-strand RNA in the initiation of synthesis of the viral genome by viral RNA-dependent RNA polymerase (RdRp). In this report, we investigated the template and binding properties of mutated and deleted RNA fragments of the 3'-end of the minus-strand HCV RNA in the presence of viral polymerase. These mutants were designed following the newly established secondary structure of this viral RNA fragment. We showed that deletion of the 3'-SL-A1 stem loop significantly reduced the level of RNA synthesis whereas modifications performed in the SL-B1 stem loop increased RNA synthesis. Study of the region encompassing the 341 nucleotides of the 3'-end of the minus-strand RNA shows that these two hairpins play a very limited role in binding to the viral polymerase. On the contrary, deletions of sequences in the 5'-end of this fragment greatly impaired both RNA synthesis and RNA binding. Our results strongly suggest that several domains of the 341 nucleotide region of the minus-strand 3'-end interact with HCV RdRp during in vitro RNA synthesis, in particular the region located between nucleotides 219 and 239.