**2.** *Citrus tristeza virus* **genome structure, organization and gene function**

The CTV RNA genome structure resembles that of *Coronaviruses,* and it is organized in twelve open reading frames (ORFs) and two non-translated regions (NTR) at the 5´ and 3´ terminus (Figure 2) [2]. The 5´ termini of the CTV genome is protected with a cap structure [2]. The 5' NTR of around 107 nt contains the sequences necessary for both replication and particle assembly [6, 23, 24]. Remarkably, the CTV 5´NTR predicted secondary structure is similar even for divergent genotypes and folded in two stem-loops separated by a short spacer region [23, 25]. The 3´NTR (273 nt) lacks a poly-A tract and does not appear to fold in a tRNA-like structure [2] but instead consists a predicted secondary structure of minimum energy of 10 stem-loop (SL) structures [26].

The study of the CTV genetics and the virus-host interactions have been hampered during long time as a consequence of the difficulties of experimenting with a virus with a large RNA genome, assembled in fragile particles and present in reduced amounts in a tree, where CTV could take long time to colonize the entire plant and to induce symptomatology. For that reason, CTV was for decades a virus complicated to isolate and characterize. Moreover, the elevated diversity of CTV populations impeded the separation of the sequence variants, composing a specific isolate, to analyze each of the genotypes independently in order to understand every aspect of viral infection. Likewise, the myriad of diseases induced by CTV, depending on the *Citrus* host, viral strain and environmental conditions, challenged the study of the host-plant interactions. In the last century, the study of CTV genetics was focused in generating molecular techniques to improve CTV detection and genotype differentiation [5]. However, in a decade, a remarkable progress has been achieved in developing the genetic engineering tools to overcome the challenges of examining CTV genetics. A cDNA clone (*T36- CTV9*) of the Florida isolate T36 was generated and an *in vitro* genetic system was developed to analyze CTV genotypes, D-RNAs, mutants and self-replicating constructs in *Nicotiana benthamiana* protoplasts or indexing plants [6, 7, 8, 9, 10]. The last advances in CTV genetics and the different biotechnological approaches used to study CTV are discussed in this chapter.

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

**Figure 1.** Left: Viral particles from *Swinglea glutinosa (Blanco) Merr.* protoplasts, transfected with CTV isolate T36, col‐ lected at 4 dpi and examined by SSEM electron microscopy. The bar indicates 200 nm. From Albiach-Marti et al. [72]. Top right: colony of *Toxoptera citricida* (Photo: Dr. A. Urbaneja). Bottom right: *Aphis gossiipi* (Photo: Dr. A. Hermoso de

**2.** *Citrus tristeza virus* **genome structure, organization and gene function**

The CTV RNA genome structure resembles that of *Coronaviruses,* and it is organized in twelve open reading frames (ORFs) and two non-translated regions (NTR) at the 5´ and 3´ terminus (Figure 2) [2]. The 5´ termini of the CTV genome is protected with a cap structure [2]. The 5'

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The CTV genome maintains the two characteristic clusters of genes of the family *Closteroviri‐ dae* (Figure 2) [11]. The *replication gene block,* which is also conserved in the supergroup of sindbis-like viruses, comprises ORF 1a and 1b and makes up the 5´ half of the viral genome [2] (Figure 2). The ORF1a encodes a 349 kDa polyprotein with two papain-like protease domains, a type I methyltransferase-like domain, and a helicase-like domain bearing the motifs of the superfamily I helicases. The ORF1b encodes a 54 kDa protein with RNA-dependent RNA polymerase (RdRp) domains. When ORF 1 is are directly translated from the positive-strand gRNA yield a 400 kDa polyprotein [2]. The conserved *quintuple gene block* (Figure 2) is related with virion assembly and trafficking in the plant [11]. This consists of the major coat protein (CP) of 25kDa, the minor coat protein (CPm) of 27kDa and other three proteins, p61, HSP70h and p6. HSP70h is a 65 kDa protein homologue of the HSP70 plant heat-shock proteins [2], a family of plant chaperones involved in protein-protein interactions, translocation into organelles, and intracellular trafficking [12]. The p6 gene encodes a small hydrophobic protein that belongs to the single-span transmembrane proteins [2]. While CP, CPm, p61 and HSP70h are necessary for proper particle assembly, p6 is required for systemic invasion of host plant [13, 14]. The additional five ORFs located at the 3´ half of the genome (Figure 2) are the p20, an homologue of p21 of *Beet yellows virus* (BYV) (genus *Closterovirus*), and four genes encoding proteins with no homologue in other closteroviruses (p33, p18, p13 and p23) (Figure 2) [11]. The p20 protein is the main component of the CTV-induced amorphous inclusion bodies [15] and it is essential for systemic infection [14]. The multifunctional protein p23 contains a Zn finger domain that binds cooperatively both single-stranded (ss) and double-stranded (ds) RNA molecules in a non-sequence specific manner [16]. In addition, p23 controls asymmetrical accumulation of positive and negative RNA strands during viral replication, ensuring the presence of enough quantity of positive genomic RNA (gRNA) ready for virion assembly [17].

**Figure 2.** Scheme of CTV genome structure and organization. CTV ORFs are delimited by boxes. The acronyms PRO, MT, HEL and RdRp indicate protein domains of papain-like protease, methyltranferase, helicase and RNA-dependent RNA polymerase, respectively. HSP70h, CPm and CP indicate ORFs encoding a homologue of heat shock protein 70, the minor and the major coat proteins, respectively. From Karasev et al. [2].

In relation to host-plant interactions, CTV is a virus with a large genome and complex genetics, while the citrus host includes many species, varieties, and intergenic hybrids with which the virus could interact causing a range of physiological and biochemical responses. In fact, CTV evolved ending up with three proteins, CP, p20 and p23, which are suppressors of the plant RNA silencing mechanism in *N. benthamiana* and *N. tabacum* plants [18]. Unexpectedly, the ORFs that encode proteins p33, p18 and p13 are not required either for replication or assembly [6, 13] or for systemic infection of Mexican lime [*C. aurantifolia* (Christm.) Swing.] and *C. macrophylla* Wester plants [14]. Nevertheless, they are involved in CTV infection and move‐ ment in other citrus hosts [19].

39] (Figure 3). This pattern of genomic divergence was more evident between the T36-like genotypes and close relatives (groups 1 and 2) and the other five CTV groups [36]. However, two paths of sequence divergence were observed [39]. The sequence divergence between CTV genotype groups 3 to 7, although slightly increased in the 5´NTR region, was relatively constant in proportion and distribution along the genome [37, 39]. On the other hand, the T36 like genotypes and close relatives showed considerable genetic distance to the other five main CTV genotypes [36]. Actually, the comparison of the genomic sequences of T30 and T36 diverged from 5% in the 3´ NTR to as high as 58% in the 5´NTR (Figure 3) [27]. Based in these two paths of sequence divergence detected between CTV genomic sequences [39], it was speculate that the T36 genotype and relatives evolved from a recombinant of a CTV genome

The Complex Genetics of *Citrus tristeza* virus http://dx.doi.org/10.5772/56122 5

**Figure 3.** Graphic of the nucleotide identity along the CTV genome when comparing T30 genomic sequence with the

Comparison of each of the CTV regions pointed to an unevenly distributed sequence variation along the CTV genome, likely reflecting different selective pressures along the genomic RNA [26, 37, 39]. Analysis of nucleotide diversity in some coding regions between CTV strains yield values higher than 0.13. However, most of the nucleotide exchanges were reported at the third codon position, indicating the preservation of the protein sequence among divergent geno‐ types. Actually, the ratio between non-synonymous and synonymous substitutions assessed for CTV coding regions was below the value 1, thus suggesting selective pressure for amino acid conservation [40]. In addition, analysis of the CTV genomic and D-RNAs sequences indicate homologous and non-homologous recombination events among different genotypes

sequences of (A) T385 (B) T36 and (C) VT genomes. From Albiach-Marti et al. [27].

and an unknown virus millions of years ago in Asia [28].

Furthermore, several CTV genomic regions have been found to be related with viral symptom development in citrus hosts. The symptomatology determinant of SY syndrome was located at the 3´ region composed by p23 ORF and the 3´NTR [20]. Nevertheless, the p33, p18 and p13 are involved in the SP syndrome development [21], although the participation in this process of other CTV regions, undetected until the moment, has not been discarded. Mild strain cross protection has been widely applied for millions of citrus trees in Australia, Brazil and South Africa [4, 5] to protect against SP economic losses. The mechanism of this type of viral superinfection exclusion is mainly a mystery. Recently, it has been found that the lack of the functional CTV p33 protein completely eliminated the ability of the virus to exclude superin‐ fection by the same or closely related virus [22].
