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Journal of Virology

Siddarame Gowda, María A. Ayllón, Tatineni Satyanarayana, William O. Dawson - Citrus Research and Education Center, University of Florida, Lake Alfred, Florida 33850

Citrus tristeza virus (CTV) produces more than thirty 3′- or 5′-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5′-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3′ genes deleted produce only the larger LMT1 RNAs. These 5′-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5′TR) surrounding the putative termination sites (nt ∼550 to 1000) was duplicated in the 3′ end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5′-terminal plus-strand sgRNA, here much larger (∼11 kb), apparently by termination. Surprisingly, a new 3′-terminal sgRNA was observed from the duplicated 5′TR. A large 3′-terminal sgRNA resulting from the putative promoter activity of the native 5′TR was not observed, possibly because of the down-regulation of a promoter ∼19 kb from the 3′ terminus. However, we were able to observe a sgRNA produced from the native 5′TR of a small defective RNA, which placed the native 5′TR closer to the 3′ terminus, demonstrating sgRNA promoter activity of the native 5′TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5′TR (in the 3′ position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3′- and 5′-terminal sgRNAs. These mutations, when introduced in frame in the native 5′TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3′-terminal sgRNA while having no impact on replication, demonstrating that neither 5′- nor 3′-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5′TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3′ open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3′-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses.

Citrus tristeza virus (CTV), a member of the genus Closterovirus of the family Closteroviridae, consists of a 19.3-kb single-stranded RNA genome of positive polarity organized into 12 open reading frames (ORFs) (9, 15). The virion RNA serves as a messenger to produce two polyproteins: one contains two papain-like proteases (L-Pro) plus methyltransferase-like (MTR) and helicase-like (HEL) domains, and the larger polyprotein has an additional RNA-dependent RNA polymerase-like domain that is thought to result from a +1 frameshift (9). The remaining 10 ORFs are expressed through a nested set of 3′-coterminal subgenomic RNAs (sgRNAs) (6). Only the 5′ ORFs 1a and 1b are required for replication in protoplasts (19), while the early-expressed 3′-most gene (p23) (14) is required for asymmetrical accumulation of plus and minus strands of both genomic and sgRNAs (20).

In infected cells, CTV produces an unusually large diversity of RNAs. In addition to the expected genomic RNA and its complementary copy, each 3′ ORF controller element produced three different RNAs: a 3′-terminal plus-strand RNA to serve as messenger for the adjacent ORF and a relatively large amount of a corresponding minus-strand sgRNA, plus a 5′-terminal plus-strand sgRNA, apparently produced by termination near the controller element during genomic RNA synthesis (4). Considering the 9 or 10 3′ controller elements (only 9 3′-terminal sgRNAs are visible from Northern blot hybridizations), this sums to 29 to 32 RNA species.

In addition, among the most abundant RNAs produced by CTV are two small 5′-coterminal plus-strand sgRNAs of ∼800 nucleotides (nt) (2, 12). These RNAs, designated low-molecular-weight tristeza (LMT) RNAs (12), are populations with heterogenous 3′ termini at nt 842, 854 (LMT1) and nt 744 to 746 (LMT2) for the VT strain of CTV (2). Since there were no corresponding minus-strand sgRNAs complementary to the LMT RNAs, it was proposed that the latter were produced by premature termination during genomic RNA synthesis (2, 12). All of the several different strains of wild-type CTV examined in protoplasts produced both populations of LMT RNAs, yet the CTV replicon with all of the 3′ ORFs deleted (19) produced only the larger LMT1 RNAs (2). Similar 5′-terminal plus-strand sgRNAs have been observed for other members of the Closteroviridae: a similarly sized RNA for Beet yellows virus (BYV), a related Closterovirus (5), and a larger RNA for Lettuce infectious yellows virus, a Crinivirus (18).

In the present study, we characterized a cis-acting element (the 5′ termination region [5′TR]) in the area corresponding to the 3′ termini of the LMT1 RNAs that effects the production of these sgRNAs. The sequences surrounding the putative LMT1 termination region were located within the replicase polypeptide coding region and could not be extensively manipulated without interfering with virus replication. We overcame this limitation by duplicating this region into the 3′ end of the genome of the CTV replicon, where the cis-acting element continued to function, producing a much longer 5′-terminal plus-strand RNA. Unexpectedly, this cis-acting element functioned as a strong sgRNA promoter when positioned near the 3′ terminus, inducing the accumulation of high levels of a new 3′-terminal plus-strand sgRNA. Mapping and characterization of the duplicated terminator/promoter element allowed precise mutagenesis of the 5′TR at its native location without affecting the replicase function. Remarkably, mutation of the native cis-acting element, such that neither the 5′-terminal nor the 3′-terminal sgRNAs were produced, failed to detectably reduce the replication of CTV in protoplasts.

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Transcription Strategy in a Closterovirus: a Novel 5′-Proximal Controller Element of Citrus Tristeza Virus Produces 5′- and 3′-Terminal Subgenomic RNAs and Differs from 3′ Open Reading Frame Controller Elements
77

Siddarame Gowda, María A. Ayllón, Tatineni Satyanarayana, William O. Dawson - Citrus Research and Education Center, University of Florida, Lake Alfred, Florida 33850

Transcription Strategy in a Closterovirus: a Novel 5′-Proximal Controller Element of Citrus Tristeza Virus Produces 5′- and 3′-Terminal Subgenomic RNAs and Differs from 3′ Open Reading Frame Controller Elements

Citrus tristeza virus (CTV) produces more than thirty 3′- or 5′-terminal subgenomic RNAs (sgRNAs) that accumulate to various extents during replication in protoplasts and plants. Among the most unusual species are two abundant populations of small 5′-terminal sgRNAs of approximately 800 nucleotides (nt) termed low-molecular-weight tristeza (LMT1 and LMT2) RNAs. Remarkably, CTV replicons with all 10 3′ genes deleted produce only the larger LMT1 RNAs. These 5′-terminal positive-sense sgRNAs do not have corresponding negative strands and were hypothesized to be produced by premature termination during plus-strand genomic RNA synthesis. We characterized a cis-acting element that controls the production of the LMT1 RNAs. Since manipulation of this cis-acting element in its native position (the L-ProI region of replicase) was not possible because the mutations negatively affect replication, a region (5′TR) surrounding the putative termination sites (nt ∼550 to 1000) was duplicated in the 3′ end of a CTV replicon to allow characterization. The duplicated sequence continued to produce a 5′-terminal plus-strand sgRNA, here much larger (∼11 kb), apparently by termination. Surprisingly, a new 3′-terminal sgRNA was observed from the duplicated 5′TR. A large 3′-terminal sgRNA resulting from the putative promoter activity of the native 5′TR was not observed, possibly because of the down-regulation of a promoter ∼19 kb from the 3′ terminus. However, we were able to observe a sgRNA produced from the native 5′TR of a small defective RNA, which placed the native 5′TR closer to the 3′ terminus, demonstrating sgRNA promoter activity of the native 5′TR. Deletion mutagenesis mapped the promoter and the terminator activities of the 5′TR (in the 3′ position in the CTV replicon) to a 57-nt region, which was folded by the MFOLD computer program into two stem-loops. Mutations in the putative stem-loop structures equally reduced or prevented production of both the 3′- and 5′-terminal sgRNAs. These mutations, when introduced in frame in the native 5′TR, similarly abolished the synthesis of the LMT1 RNAs and presumably the large 3′-terminal sgRNA while having no impact on replication, demonstrating that neither 5′- nor 3′-terminal sgRNA is necessary for replication of the replicon or full-length CTV in protoplasts. Differences between the 5′TR, which produced two plus-strand sgRNAs, and the cis-acting elements controlling the 3′ open reading frames, which produced additional minus-strand sgRNAs corresponding to the 3′-terminal mRNAs, suggest that the different sgRNA controller elements had different origins in the modular evolution of closteroviruses.

Citrus tristeza virus (CTV), a member of the genus Closterovirus of the family Closteroviridae, consists of a 19.3-kb single-stranded RNA genome of positive polarity organized into 12 open reading frames (ORFs) (9, 15). The virion RNA serves as a messenger to produce two polyproteins: one contains two papain-like proteases (L-Pro) plus methyltransferase-like (MTR) and helicase-like (HEL) domains, and the larger polyprotein has an additional RNA-dependent RNA polymerase-like domain that is thought to result from a +1 frameshift (9). The remaining 10 ORFs are expressed through a nested set of 3′-coterminal subgenomic RNAs (sgRNAs) (6). Only the 5′ ORFs 1a and 1b are required for replication in protoplasts (19), while the early-expressed 3′-most gene (p23) (14) is required for asymmetrical accumulation of plus and minus strands of both genomic and sgRNAs (20).

In infected cells, CTV produces an unusually large diversity of RNAs. In addition to the expected genomic RNA and its complementary copy, each 3′ ORF controller element produced three different RNAs: a 3′-terminal plus-strand RNA to serve as messenger for the adjacent ORF and a relatively large amount of a corresponding minus-strand sgRNA, plus a 5′-terminal plus-strand sgRNA, apparently produced by termination near the controller element during genomic RNA synthesis (4). Considering the 9 or 10 3′ controller elements (only 9 3′-terminal sgRNAs are visible from Northern blot hybridizations), this sums to 29 to 32 RNA species.

In addition, among the most abundant RNAs produced by CTV are two small 5′-coterminal plus-strand sgRNAs of ∼800 nucleotides (nt) (2, 12). These RNAs, designated low-molecular-weight tristeza (LMT) RNAs (12), are populations with heterogenous 3′ termini at nt 842, 854 (LMT1) and nt 744 to 746 (LMT2) for the VT strain of CTV (2). Since there were no corresponding minus-strand sgRNAs complementary to the LMT RNAs, it was proposed that the latter were produced by premature termination during genomic RNA synthesis (2, 12). All of the several different strains of wild-type CTV examined in protoplasts produced both populations of LMT RNAs, yet the CTV replicon with all of the 3′ ORFs deleted (19) produced only the larger LMT1 RNAs (2). Similar 5′-terminal plus-strand sgRNAs have been observed for other members of the Closteroviridae: a similarly sized RNA for Beet yellows virus (BYV), a related Closterovirus (5), and a larger RNA for Lettuce infectious yellows virus, a Crinivirus (18).

In the present study, we characterized a cis-acting element (the 5′ termination region [5′TR]) in the area corresponding to the 3′ termini of the LMT1 RNAs that effects the production of these sgRNAs. The sequences surrounding the putative LMT1 termination region were located within the replicase polypeptide coding region and could not be extensively manipulated without interfering with virus replication. We overcame this limitation by duplicating this region into the 3′ end of the genome of the CTV replicon, where the cis-acting element continued to function, producing a much longer 5′-terminal plus-strand RNA. Unexpectedly, this cis-acting element functioned as a strong sgRNA promoter when positioned near the 3′ terminus, inducing the accumulation of high levels of a new 3′-terminal plus-strand sgRNA. Mapping and characterization of the duplicated terminator/promoter element allowed precise mutagenesis of the 5′TR at its native location without affecting the replicase function. Remarkably, mutation of the native cis-acting element, such that neither the 5′-terminal nor the 3′-terminal sgRNAs were produced, failed to detectably reduce the replication of CTV in protoplasts.

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