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

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

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

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

other RNA viruses replication [17].

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 be transmitted by aphids [45].

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-RNAs [47].

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

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

**6.** *Citrus tristeza virus* **encapsidation**

mixed infections [59].

CTV assembly is unknown at the moment [52].

Differently of most elongated viruses, CTV particles are encapsidated by two different capsid proteins that coat the opposite ends of the virion [24]. About the 97% of the CTV genome is coated by CP, while the remainder 3% is coated by CPm resulting in viral particles with the emblematic tail of the members of the *Closteroviridae* family [11, 24]. The coordinate action of HSP70h and p61, in addition to the CP and CPm coat proteins, are required for proper assembly of CTV particles [13]. The previously described CTV 5´NTR conserved SL structures contains the origin of assembly of CPm, overlapping the sequences that function as a cis-acting element required for gRNA synthesis [24]. During CTV assembly, CPm begins coating the gRNA at the 5´ NTR to about nt 630. However, in the absence of HSP70h or p61, CPm may coat larger segments or even the complete gRNA. Probably, HSP70h or p61 bind to the transition zone between CP and CPm (around 630 nt) and restrict CPm to the virion tail [24]. The protein homologous to HSP70h and p61 in BYV are coordinately assembled with CPm in the virion structure and remain attached to the viral particles [58]. Although the assembly of HSP70h and p61 is not directly confirmed for CTV, RNA transcripts lacking one or both of these ORFs were unable to produce infective CTV virions [24]. Several strains with divergent genotypes could coexist in the same citrus cell. Analysis of the encapsidation of heterologous CPms in absence of HSP70h and p61 indicated reduction or lack of CPm assembly. Nevertheless, the presence of HSP70h and p61 restored CPm assembly to wild-type levels. This indicated that the HSP70h and p61 could play an important role facilitating heterologous CPm assembly in

The Complex Genetics of *Citrus tristeza* virus http://dx.doi.org/10.5772/56122 11

The region coated by CPm also overlaps the LMT2 5´ sgRNA (650 nts). Actually, LMT2 production is correlated to virion assembly since mutations in the 5´ NTR that abolish encapsidation also eliminate accumulation of LMT2. Although this represents the first evidence of a viral RNA processed by the assembly mechanism, the exact function of LMT2 in

In order to infect a plant, CTV needs to enter in the cell, and to overcome the constitutive and/or inducible plant defences, to re-program the plant cellular machinery for its viral multiplica‐ tion. The infection process will continue with the assembly of new viral particles that will move celltocellthroughtheplasmodesmata.Thisprocesswillbecompletedwiththevirallongdistance movement through the plant vascular structure to colonize systemically the plant. Each CTV gene product seems to have a primary genetic function required for the survival of the virus. However, there are secondary genetic interactions, which cause or trigger resistance or pathogenic responses in the citrus host [60, 61]. *Citrus* genus contains multitude of species, cultivars and intergenic hybrids, with which CTV could interact causing a range of physiologi‐ cal and biochemical responses. These could be from either pathogenic or asymptomatic phenotypes to limited or complete plant resistance [5]. Although most of these mechanisms are

**7.** *Citrus tristeza virus* **genetics and plant-host interactions**

**Figure 5.** Different species of D-RNAs in CTV populations. Top panel: graphic representation of a usual small CTV D-RNA compared with the CTV genome. Bottom panel: accumulation of CTV RNAs in bark extracts from sweet orange plants infected with three field isolates (1 to 3 lines), before (lines w) and after (lines T) aphid transmission. Northernblot hybridization performed using a single-stranded minus-sense riboprobes specific to the 3' end (left panel) and the 5´end (right panel) of the T36 genome. From Mawassi et al. [46] and Albiach-Marti et al. [45]

In other viral pathosystems, D-RNAs have the capacity of interfering with the viral replication process of their helper virus (named defective interfering (DI) RNAs), but those function was not reported for CTV [10]. Although their biological role is presently unknown, at least in one case, the presence of D-RNAs was suggested to modulate symptom development either increasing or decreasing CTV symptom expression [57]. Most of the CTV D-RNAs contain a complete region p23 and the 3´NTR [43] that is associated with SY development [20], thus they could have a role in symptom modulation. Therefore, it will be necessary to promote further research to elucidate the role of the D-RNAs (or DI-RNAs) in CTV replication or in modulation of pathogenic responses in the infected plant host.
