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 plus-strand 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.

FIG. 1.

(A) Schematic representation of BMV RNA3 constructs (ID RNA3) carrying repeats of sgp sequences. INT-1 and INT-2 indicate the location of the repeated sg sequence (represented by black bars) and thin lines represent the noncoding regions, while open boxes represent ORFs for 3a and for the CPs. The repeated elements [the enhancer, the poly(A) track, the core, and a downstream hairpin (5-6)] are represented below, as indicated by cut-bars at the bottom for ID1 through ID3. Vertical arrows depict the location of the mutated nucleotides in ID3. The numbers represent nucleotide positions in the wt sequence, as counted from the 5′ end. (B) The location of marker mutations in pairs of coinoculated parental ID1 to ID3 RNAs3. Solid stars, triangles, spades, and circles represent, respectively, EcoRI, HindIII, XhoI, and EcoRI. The numbers on the top show the nucleotide positions of marker mutations. (C) The predicted types of progeny RNA3-RNA3 recombinants (see also Table1) that arose due to crossover events within the INT-1 or INT-2 regions. Solid lines represent INT-1 or INT-2 regions, while open boxes represent the ORFs. The marker restriction sites are depicted, as explained above.

FIG. 2.

Northern blot analysis of the accumulation of BMV ID RNA3 constructs in C. quinoa (A) or in barley (B). The leaves were coinoculated mechanically with the in vitro-transcribed wt RNA1 and RNA2 and with ID RNA3 mutants (as indicated on the top). The progeny BMV RNA was extracted from the infected tissue, separated electrophoretically in a 1% agarose-formamide-formaldehyde gel, blotted to a nylon membrane (HybondN⁺; Amersham), and probed with a ³²P-labeled RNA complementary to the 3′ 200 nt of the plus strand. Lanes 1 to 6 (A) or 2 to 5 (B) show the RNAs extracted from the local lesion tissue of C. quinoa or of barley, respectively. The control lanes 1 and 7 show wt BMV RNA from barley. The positions of individual BMV RNA segments are indicated on the left. The histograms below show the quantification of the accumulation of sg RNA4 and RNA4′. The Northern blot autoradiograms were scanned and subjected to computer-based densitometric analysis by using the program ImageQuant 5.0 of Amersham Biosciences. The bars show the fraction (percent) by which the band intensity for RNAs 4 and 4′ contributed to the total intensity of all BMV RNA components.

FIG. 3.

Electrophoretical analysis of cDNA clones obtained during a control experiment towards testing if RT-PCR generated RNA3 recombinants. (A) The progeny BMV RNAs from separately inoculated plants (with ID2-1 or ID1-1 RNA3s) were mixed together and coamplified with RT-PCR, using the primers RW-Pst and RW-PstI. The DNA was cloned in the pUC19 vector between PstI sites, and the inserts (of 1,145 nt) were released from the resulting plasmids with PstI digestion and separated in a 1.5% agarose gel. No size variability was demonstrated among the 19 analyzed clones. (B) Exemplary restriction analysis of clones 1 and 4 from lanes 1 and 4 in panel A, respectively. Plasmid DNA was digested with the indicated restriction enzymes, and the products were separated in a 1.5% agarose gel against the 100-bp DNA standard (Promega). The size of the individual bands corresponds to the parental fragments, as predicted with the diagram (not to scale) on top.

FIG. 4.

Diagrammatic representation of a putative mechanism of crossovers at the sgp recombination hot spot. Thick lines represent minus strands of BMV RNA3, while dotted lines represent progeny plus strands. Arrows show the direction of migration of the RdRp complex (represented by a solid circle). (A) RNA-RNA crossovers during copying of plus strands of RNA3. The replicase initiates the synthesis of plus strands at the 3′ end, pauses at the sgp, and switches onto another RNA3 molecule. (B) De novo initiation of RNA4 transcription.