A Transcriptionally Active Subgenomic Promoter Supports Homologous Crossovers in a Plus-Strand RNA Virus

Abstract

Genetic RNA recombination plays an important role in viral evolution, but its molecular mechanism is not well understood. In this work we describe homologous RNA recombination activity that is supported by a subgenomic promoter (sgp) region in the RNA3 segment of brome mosaic bromovirus (BMV), a tripartite plusstrand RNA virus. The crossover frequencies were determined by coinoculations with pairs of BMV RNA3 variants that carried a duplicated sgp region flanked by marker restriction sites. A region composed of the sgp core, a poly(A) tract, and an upstream enhancer supported homologous exchanges in 25% of the analyzed RNA3 progeny. However, mutations in the sgp core stopped both the transcription of the sgp RNA and homologous recombination. These data provide evidence for an association of RNA recombination with transcription.Genetic recombination is an important process leading to diversity among living organisms. The mechanisms of crossing over (general homologous recombination) and other routes of genetic exchange are relatively well studied in DNA-based organisms. In contrast, although RNA recombination has been studied extensively for several RNA virus groups, including bromoviruses, picornaviruses, coronaviruses, tombusviruses, and bacteriophages (8), its mechanism is not well understood (8, 13). Brome mosaic bromovirus (BMV) is a tripartite RNA virus where RNA components 1 and 2 encode, respectively, the replicase proteins 1a and 2a, while RNA3 encodes the movement (3a) and the coat proteins (CPs) (3). CP is expressed from a subgenomic (sg) RNA4.The subgenomic promoter (sgp) for RNA4 is located internally on the minus strand of BMV RNA3. It includes a core region that is responsible for binding of the viral RNA polymerase (RdRp) and the initiation of transcription (30), an "enhancing" region, a poly(A) stretch, and a downstream portion (21, 34). Besides BMV, the multielement nature of sgp's has been demonstrated in other RNA viruses (24).There is only limited information about recombination events in natural populations of viral RNA molecules. Recombination hot spots were observed during the course of infection in poliovirus (16) and in human immunodeficiency virus type 1 (36). It has been proposed that recombination within the sg RNA start sites has led to the formation of genera of luteoviruses (23) or has served as a factor for the modular exchange and rearrangements of the genomes of closteroviruses (5). Also, the recombination hot spots are thought to be associated with RNA replication enhancers, such as those found in turnip crinkle carmovirus (9, 30). RNA structure has been reported to play a role in promoting RNA crossovers in retroviruses (4).In BMV, the frequency of homologous intersegmental crossovers is approximately 10 times higher than that of nonhomologous crossovers (27). A model of the replicase template switching has been proposed to explain the observed homologous crossovers between BMV RNAs (28, 29). An increased recombination activity has been demonstrated within the intercistronic r...

The AU-Rich RNA Recombination Hot Spot Sequence of Brome Mosaic Virus Is Functional in Tombusviruses: Implications for the Mechanism of RNA Recombination

“…8). A different class of cis-acting element, namely, the subgenomic promoter region, has also been proposed to promote RNA recombination in luteoviruses (10a) and BMV (34). In summary, the discovery of the role of the RII(Ϫ) element in RNA recombination, in combination with published data cited above, suggests that cis-acting elements might play much wider roles in viral RNA recombination than previously anticipated.…”

Dissecting the Requirement for Subgenomic Promoter Sequences by RNA Recombination of Brome Mosaic Virus In Vivo: Evidence for Functional Separation of Transcription and Recombination

“…Figure 1 shows the sequences of ID RNA3 constructs that carry insert repeats of the sgp sequence at the INT-2 locus. The repeats were introduced downstream of the CP open reading frame (ORF) (at the INT-2 locus) by ligation of PCR-amplified (from the original wt pB3TP7 plasmid [28]) cDNA products at the BamHI site of plasmid SF-25 (11,54). In several cases the constructs were obtained by sequential PCR, using the product of the first PCR as a primer for the second PCR.…”

“…4). To determine how sgp sequence modifications affected BMV RNA accumulation and INT-2 transcription activity, the progeny RNAs were harvested at the time that secured an equal accumulation of the virus for all of the constructs, i.e., 10 days postinfection, as established previously (54). The leaf tissues were quickly frozen in liquid nitrogen immediately after harvesting to prevent RNA degradation, and then the total RNAs were extracted from equal weights (100 mg) of the combined local lesion tissue by using a standard RNA isolation protocol.…”

“…Detection of crossovers was possible by coinfection with pairs of RNA3 variants carrying marker mutations at different positions. Further work has pinpointed the crossovers to the sgp sequence, and it was also observed that RNA recombination and transcription events overlapped within the sgp (11,15,19,54). The sgp is recognized by the replicase enzyme that binds internally to minus strands (46) and synthesizes the sg RNA4.…”

Sequenciamento e análise da variabilidade genética de vírus transmitidos por ácaros do gênero <i>Brevipalpus </i>no Brasil

“…Esses resultados contrastantes nos levou a avaliar possíveis eventos de recombinação no RNA2 de CiLV-C. O RNA sub-genômico específico para o gene p61 foi detectado durante a replicação de CiLV-C e CiLV-C2 (PASCON et al, 2006;ROY et al, 2013b). A RI do RNA2 dos cilevirus encontra-se anterior a p61 e provavelmente abriga elementos promotores necessários para dirigir a expressão do RNA sub-genômico da p61, essa associação de recombinação de RNA com a transcrição foi observada em outros vírus com esse sistema de expressão, como em Brome mosaic virus (WIERZCHOSLAWSKI et al, 2003(WIERZCHOSLAWSKI et al, , 2004). …”