**4.** *Citrus tristeza virus* **replication and gene expression**

CTV replication is an extraordinary process that generates at least 35 different species of viral RNA in CTV-infected cells (Figure 4) [44] plus a myriad of D-RNAs [45, 46, 47] (Figure 5). The viral genomic sequences necessary for CTV replication are the *replication gene block* plus the 3´ and 5´NTRs, which contain the cis-acting elements indispensable forthis process (Figure 4). In fact, a T36 CTV replicon consisting in only these genomic regions is able to selfreplicate in protoplasts of *N. benthamiana* [6]. As indicated previously, the CTV 5´NTR predicted secondary structure folded into two SL separated by a short spacer region [25]. Directed mutations disrupting this predicted secondary structure were shown to abolish replication, whereas compensatory mutations resumed replication, suggesting that the secondary structure of the 5´ NTR is more important than the primary structure for CTV replication [23]. Conversely, the basic function of the 3´ NTR (273 nt) is minus-strand initiation for the CTV gRNA and the subgenomic (sg) RNAs [26]. The 3´NTR consists in a predicted secondary structure of 10 SL structures. While the core of the 3' replication signal was located in the primary structure of three of the central stem-loops (SL4, SL6 and SL8), the secon‐ dary structure of the other stem-loops (SL3, SL5, SL7 and SL9) proved dispensable but required for efficient replication [26]. In addition, all CTV genomes retain a CCA triplet at the 3´ termini necessary to initiate replication [26].

**Figure 4.** RNA species produced *in cis* during CTV replication. Main panel: Scheme of the different CTV RNA species. Black lines: single-stranded positive-sense RNAs. Grey lines: single-stranded negative-sense RNAs. The acronyms gRNA, sgRNA and LMT RNA indicate genomic, subgenomic and *low molecular-weight tristeza* RNAs, respectively. The signs (+) and (-) specify plus and minus-strand RNAs, respectively. Black arrows on the CTV gRNA(-) line designate the ap‐ proximate position of the CTV controller elements in the CTV genome. Small left panel: Accumulation of T36 double strain (ds) RNAs in citrus plants showing the 3´coterminal sgRNAs produced during replication and expression of the ten 3´half ORFs of the CTV genome. Northern-blot hybridization performed using a single-stranded negative-sense riboprobes specific to the 3' end of T36 genomic sequence. From Karasev et al. [2, 50], Gowda et al. [44, 51, 52] and

The Complex Genetics of *Citrus tristeza* virus http://dx.doi.org/10.5772/56122 7

In the first step of the CTV genome replication, the viral replicase uses the single stranded positive-sense gRNA (CTV gRNA (+)) from the uncoated viral particles, as template to generate a homologous single-stranded negative –sense CTV gRNA (CTV gRNA (-)) (Figure 4). The CTV gRNA(-) molecules will function as basis for the synthesis of the CTV progeny of positivestrand gRNAs. The CTVgRNA(+) molecules would act as RNA messenger for expression of viral proteins or as a pool of CTV gRNAs ready to be incorporated into virions to produce newly infectious viral particles. The new CTV gRNA(+) could also serve as template for the

synthesis of fresh CTVgRNA(-) molecules to start all over the process [44].

Ayllon et al. [49, 48].

Wild CTV populations could be composed by divergent genotypes [3]. In the case of mixed infections in the same plant cell, it is essential to determine whether a specific replicase complex is able to recognize the cis–acting elements of the 3´or 5´ NTR of other genomic variants. The exchange of 5´NTR and 3´NTR sequences, from different main genotypes, into the *T36-CTV9* infectious clone decreases replication as the degree of sequence divergence increases. There‐ fore, indicating partial compatibility of the T36 replicase complex with diverged 5´ and 3´ cisacting elements, thus suggesting limited heterologous replication in mixed viral infections [6].

[30, 32, 41, 42, 43], possibly as a result of mixed infections on trees that are recurrently inocu‐

Conversely, in spite of this genetic variability, sequence comparisons of some CTV ge‐ nomes revealed a remarkable viral genetic stasis as the genomes of some CTV strains, separated geographically and in time, were found essentially identical [27]. This genetic stability has been explained as a consequence of strong selection and competition between the mutants that arise in each replication cycle, which creates equilibrium in the viral quasispecies distribution [27]. In this context, there is a hypothesis to explain the high sequence variability found in the wild CTV isolates [3, 27]. In a fist stage, each of the main genotypes evolved separately in different *Citrus* species at their point of origin in Asia. This was followed by the dispersal of the main CTV genotypes to different environments around the world with the advent of the modern citrus industry in the XIX century. After that, RNA virus muta‐ tion, due the error-prone nature of RNA-dependent RNA polymerases, in addition to recombination events between diverged sequence variants, plus selection, genetic drift and

CTV replication is an extraordinary process that generates at least 35 different species of viral RNA in CTV-infected cells (Figure 4) [44] plus a myriad of D-RNAs [45, 46, 47] (Figure 5). The viral genomic sequences necessary for CTV replication are the *replication gene block* plus the 3´ and 5´NTRs, which contain the cis-acting elements indispensable forthis process (Figure 4). In fact, a T36 CTV replicon consisting in only these genomic regions is able to selfreplicate in protoplasts of *N. benthamiana* [6]. As indicated previously, the CTV 5´NTR predicted secondary structure folded into two SL separated by a short spacer region [25]. Directed mutations disrupting this predicted secondary structure were shown to abolish replication, whereas compensatory mutations resumed replication, suggesting that the secondary structure of the 5´ NTR is more important than the primary structure for CTV replication [23]. Conversely, the basic function of the 3´ NTR (273 nt) is minus-strand initiation for the CTV gRNA and the subgenomic (sg) RNAs [26]. The 3´NTR consists in a predicted secondary structure of 10 SL structures. While the core of the 3' replication signal was located in the primary structure of three of the central stem-loops (SL4, SL6 and SL8), the secon‐ dary structure of the other stem-loops (SL3, SL5, SL7 and SL9) proved dispensable but required for efficient replication [26]. In addition, all CTV genomes retain a CCA triplet at the

Wild CTV populations could be composed by divergent genotypes [3]. In the case of mixed infections in the same plant cell, it is essential to determine whether a specific replicase complex is able to recognize the cis–acting elements of the 3´or 5´ NTR of other genomic variants. The exchange of 5´NTR and 3´NTR sequences, from different main genotypes, into the *T36-CTV9* infectious clone decreases replication as the degree of sequence divergence increases. There‐ fore, indicating partial compatibility of the T36 replicase complex with diverged 5´ and 3´ cisacting elements, thus suggesting limited heterologous replication in mixed viral infections [6].

gene flow could have been promoted rapid evolution [3, 5, 27].

6 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

3´ termini necessary to initiate replication [26].

**4.** *Citrus tristeza virus* **replication and gene expression**

lated by aphid transmission.

**Figure 4.** RNA species produced *in cis* during CTV replication. Main panel: Scheme of the different CTV RNA species. Black lines: single-stranded positive-sense RNAs. Grey lines: single-stranded negative-sense RNAs. The acronyms gRNA, sgRNA and LMT RNA indicate genomic, subgenomic and *low molecular-weight tristeza* RNAs, respectively. The signs (+) and (-) specify plus and minus-strand RNAs, respectively. Black arrows on the CTV gRNA(-) line designate the ap‐ proximate position of the CTV controller elements in the CTV genome. Small left panel: Accumulation of T36 double strain (ds) RNAs in citrus plants showing the 3´coterminal sgRNAs produced during replication and expression of the ten 3´half ORFs of the CTV genome. Northern-blot hybridization performed using a single-stranded negative-sense riboprobes specific to the 3' end of T36 genomic sequence. From Karasev et al. [2, 50], Gowda et al. [44, 51, 52] and Ayllon et al. [49, 48].

In the first step of the CTV genome replication, the viral replicase uses the single stranded positive-sense gRNA (CTV gRNA (+)) from the uncoated viral particles, as template to generate a homologous single-stranded negative –sense CTV gRNA (CTV gRNA (-)) (Figure 4). The CTV gRNA(-) molecules will function as basis for the synthesis of the CTV progeny of positivestrand gRNAs. The CTVgRNA(+) molecules would act as RNA messenger for expression of viral proteins or as a pool of CTV gRNAs ready to be incorporated into virions to produce newly infectious viral particles. The new CTV gRNA(+) could also serve as template for the synthesis of fresh CTVgRNA(-) molecules to start all over the process [44].

Another function of the CTV gRNA (-) is to serve as template to produce high quantities of single and double strain sgRNAs during the expression mechanism of the ten ORFs situated at the 3´half of the genome (Figure 4 and 5) [44, 48, 49, 50]. Unlike the large animal viruses of the *Nidovirales*, the 3´ sgRNAs of CTV do not share a common 5´ terminus and the sgRNA transcription mechanism resembles the transcriptional mechanism of other Sindbis-like viruses [50]. The synthesis of each 3´ coterminal sgRNA is controlled by its corresponding cisacting element (controller element (CE)) (Figure 4). Probably each CTV CE could act as promoter or terminator of the CTV RNAs during the replication process [44, 48, 49, 50]. However, if the CEs function as internal promoters for the generation of positive-strand sgRNAs, using as template the CTVgRNA(-) molecules (Figure 4), or act as terminators for the synthesis of negative-strand gRNAs (by premature termination at the CE site), or both, is still unclear [44]. In addition to the plus and minus- sense 3´coterminal sgRNAs, the CEs corre‐ sponding to each of the ten 3´ORFs produce a reduced amount of a set of 5´coterminal positivestrand sgRNAs (Figure 4), probably due to premature termination during the synthesis of the CTV gRNA(+) [44]. Moreover, CTV generates significant amounts of *low molecular-weight tristeza* (LMT1 and LMT2), two positive-strand 5´co-terminal sgRNAs population with heterogeneous 3´termini at nt 842-854 and 744-746, respectively (Figure 4 and 5) [46, 47]. LMT 1 and LMT 2 are generated and accumulated differently [51, 52]. LMT1 is likely created by premature termination during CTV gRNA(+) synthesis at a 5´ CE situated in the PRO I domain of the replicase (Figure 4). This 5´ CE acts as a strong promoter when placed immediately upstream of the ORFs near the 3´ terminus [51]. In contrast of the 3´ CEs, which are able to generate plus and minus-strand sgRNAs, the 5´ CE of the LMT 1 only promoted the synthesis of positive-strand sgRNAs (Figure 4) [51]. In fact, the RNA termination and initiation sites of the 5' CE, compared to those of 3' CEs, occur at opposite ends of the corresponding minimal active CE site [49]. Therefore, as a result of the replication process, CTV produces high amounts of viral RNA species in the infected cell (Figure 4). The total (gRNAs plus sgRNAs) positive to negative-strand RNA ratio (approximately 40 to 50:1) falls within the range of the genomic RNAs of most positive-strand RNA viruses, particularly the more similar alphavirus super‐ group and large complex viruses of the *Nidovirales* [26]. However, during CTV replication, only the positive-strand gRNA accumulates approximately 10 to 20 times more than their negative-strand gRNA homologues, a rather lower ratio compared to those generated during other RNA viruses replication [17].

expression of each of the ten 3' proximal ORFs is regulated independently both in amount and

The Complex Genetics of *Citrus tristeza* virus http://dx.doi.org/10.5772/56122 9

In addition to the 35 different species of RNA created during replication, CTV could accumu‐ late considerable amounts of D-RNAs in infected cells (Figure 5) [46]. CTV D-RNAs vary in size, abundance and sequence [41, 45, 46] and could be encapsidated into particles and could

Generally, D-RNAs bear a genome from 2.0 to 5.0 kb and are composed by variable portions of the 3' and 5' termini of CTV genomic RNA with large internal deletions (Figure 5). Never‐ theless, some D-RNAs comprising the two termini and a non-contiguous internal sequence or a non-viral sequence, plus large D-RNAs of 10-12 kb including in their 5' proximal region the ORFs 1a and 1b, or with a 3' region homologous to the ten CTV 3' terminal ORFs, have been described [41, 43, 46, 55]. These large D-RNAs resembled the RNAs 1 and 2 distinctive of the bipartite *Criniviruses,* also included in the *Closteroviridae* family. Moreover, the D-RNA containing the complete CTV replicase constitutes a novel class of large self-replicating D-

CTV D-RNAs characteristic genomic structure suggests an origin in the recombination events during viral replication. In this way, some large D-RNA bear a 5´ termini identical o slightly larger than the 5´ sgRNA generated by the CE of the p33 ORF [47], and the small ones usually contain a 3´ termini identical to 3´ sgRNA of p23 ORF [43]. Additionally, a repeated 4-5 nt, (corresponding to two CTV genomic regions) was reported flanking the D-RNA 3´ and 5´ termini junction sites indicating that D-RNAs are probably created during the generation of

D-RNAs require the viral machinery for their survival. The D-RNA replication *in trans* was examined using infectious D-RNAs and the *in vitro* genetic system of *T36/CTV9* [10, 6]. The minimal D-RNA sequence required for replication are a 5´ proximal region of 1kb and a 3´ termini limited to the CTV 3´NTR. In addition, efficient replication of D-RNAs involves some spacing between these terminal cis-acting signals and a continuous ORF through most of the 5´ proximal regions of the D-RNA sequence [10, 56]. CTV field isolates are composed by viral populations of divergent genotypes. In this case, an important point is to understand the dynamics of generation and accumulation of D-RNAs in a specific plant cell infected with distinct CTV genotypes. Mawassi et al., [56] demonstrate that some wild-type populations of CTV are capable of supporting the replication of synthetic divergent D-RNAs. However, replacement of 5´ region (which is the most variable among CTV strains) of a particular synthetic D-RNA, with the corresponding sequence from different main CTV genotypes, resulted in chimeric D-RNAs that were replicated to detectable levels by some CTV genotypes but not with the others. Consequently, differential specificities of distinct CTV replicase complexes with divergent D-RNA replication signals are possibly affecting the maintenance

the positive-strand sgRNA or gRNA by a template-switching mechanism [41, 43].

of D-RNA population structures in the infected plant cell.

timing [46, 47].

RNAs [47].

**5.** *Citrus tristeza virus* **defective RNAs**

be transmitted by aphids [45].

The expression of the CTV genome, which potentially yields at least nineteen protein products, resembles that of *Coronaviruses* [50]. This remarkable process includes at least three different RNA expression mechanisms widely used by positive-strand RNA viruses: proteolytic processing of the polyprotein precursor, translational frameshifting and the generation of a nested set of ten 3'-coterminal sgRNAs [50]. Therefore, the ORFs 1a and 1b are directly translated, from the positive-strand gRNA, to yield a 400 kDa polyprotein that is later proteolytically processed in, at least, nine protein products. The ORF1b encodes a 54 kDa protein with RdRp domains that is occasionally translated after ORF 1a by a +1 ribosomal frameshifting [2]. Additionally, as indicated above, the 10 ORFs located at the 3' half of the CTV genome are expressed by the synthesis of ten 3' co-terminal sgRNAs (Figure 4). Each 3' sgRNA serve as RNA messenger for the translation of its 5' proximal ORF [13, 46] and the expression of each of the ten 3' proximal ORFs is regulated independently both in amount and timing [46, 47].
