Citrus tristeza virus co-opts glyceraldehyde 3-phosphate dehydrogenase for its infectious cycle by interacting with the viral-encoded protein p23.

KEY MESSAGE: Citrus tristeza virus encodes a unique protein, p23, with multiple functional roles that include co-option of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase to facilitate the viral infectious cycle. The genome of citrus tristeza virus (CTV), genus Closterovirus family Closteroviridae, is a single-stranded (+) RNA potentially encoding at least 17 proteins. One (p23), an RNA-binding protein of 209 amino acids with a putative Zn-finger and some basic motifs, displays singular features: (i) it has no homologues in other closteroviruses, (ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and (iii) it mediates asymmetric accumulation of CTV RNA strands, intracellular suppression of RNA silencing, induction of some CTV syndromes and enhancement of systemic infection when expressed as a transgene ectopically or in phloem-associated cells in several Citrus spp. Here, a yeast two-hybrid screening of an expression library of Nicotiana benthamiana (a symptomatic experimental host for CTV), identified a transducin/WD40 domain protein and the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potential host interactors with p23. Bimolecular fluorescence complementation corroborated the p23-GAPDH interaction in planta and showed that p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH in the cytoplasm (forming aggregates) and in plasmodesmata. The latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two others affecting the Zn-finger and one internal basic motif. Virus-induced gene silencing of GAPDH mRNA resulted in a decrease of CTV titer as revealed by real-time RT-quantitative PCR and RNA gel-blot hybridization. Thus, like other viruses, CTV seems to co-opt GAPDH, via interaction with p23, to facilitate its infectious cycle.

Functional diversification upon leader protease domain duplication in the Citrus tristeza virus genome: Role of RNA sequences and the encoded proteins.

Viruses from the family Closteroviridae show an example of intra-genome duplications of more than one gene. In addition to the hallmark coat protein gene duplication, several members possess a tandem duplication of papain-like leader proteases. In this study, we demonstrate that domains encoding the L1 and L2 proteases in the Citrus tristeza virus genome underwent a significant functional divergence at the RNA and protein levels. We show that the L1 protease is crucial for viral accumulation and establishment of initial infection, whereas its coding region is vital for virus transport. On the other hand, the second protease is indispensable for virus infection of its natural citrus host, suggesting that L2 has evolved an important adaptive function that mediates virus interaction with the woody host.

A 5'-proximal region of the Citrus tristeza virus genome encoding two leader proteases is involved in virus superinfection exclusion.

Superinfection exclusion (SIE), a phenomenon in which a primary virus infection prevents a secondary infection with the same or closely related virus, has been observed with various viruses. Earlier we demonstrated that SIE by Citrus tristeza virus (CTV) requires viral p33 protein. In this work we show that p33 alone is not sufficient for virus exclusion. To define the additional viral components that are involved in this phenomenon, we engineered a hybrid virus in which a 5'-proximal region in the genome of the T36 isolate containing coding sequences for the two leader proteases L1 and L2 has been substituted with a corresponding region from the genome of a heterologous T68-1 isolate. Sequential inoculation of plants pre-infected with the CTV L1L2T68 hybrid with T36 CTV resulted in superinfection with the challenge virus, which indicated that the substitution of the L1-L2 coding region affected SIE ability of the virus.

The RNA-dependent RNA polymerase of Citrus tristeza virus forms oligomers.

The RNA-dependent RNA polymerases (RdRp) from Citrus tristeza virus (CTV) were tagged with HA and FLAG epitopes. Differentially tagged proteins were expressed either individually or concomitantly in Escherichia coli. Immunoprecipitation of the expressed proteins with anti-FLAG antibody followed by Western blot with anti-HA antibody demonstrated that molecules of RdRp from CTV interact to form oligomers. Yeast two-hybrid assays showed that molecules of RdRp interact in eukaryotic cells. Co-immunoprecipitation with anti-FLAG antibody of truncated HA-tagged RdRps (RdRpΔ1-166-HA, RdRpΔ1-390-HA, RdRp1-169-HA) co-expressed with full-length RdRp-FLAG showed that only RdRp1-169-HA interacted with the full-length FLAG-RdRp. Yeast two-hybrid assays with truncated RdRp constructs confirmed that the oligomerization site resides in the N-terminal region and that the first 169 aa of CTV RdRp are necessary and sufficient for oligomerization both in bacterial and yeast cells. Development of control strategies targeting viral RdRp oligomer formation may inhibit virus replication and prove useful in control of CTV.

In vivo and in vitro expression analysis of the RNA-dependent RNA polymerase of Citrus tristeza virus.

Expression of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) was studied in vivo and in vitro using a polyclonal antiserum raised against the recombinant CTV-RdRp protein. Although a 57-kDa CTV-RdRp was expected to be expressed by a +1 translational frameshift at the carboxyl terminus of a 400-kDa polyprotein, a 50-kDa protein was detected in CTV-infected but not in healthy citrus tissue by Western blot. This suggests that the RdRp was cleaved from the CTV polyprotein. The 50-kDa protein was present in both the cytoplasmic and membrane fractions, but it accumulated mainly in the membrane fraction, where most of the replication-associated proteins of RNA viruses are found. When the expression of a cloned CTV-RdRp gene encoding a 60-kDa fusion protein was studied in vitro in a rabbit reticulocyte lysate system, two smaller proteins of about 50 kDa and 10 kDa were detected in addition to the expected 60-kDa protein. All three proteins were immunoprecipitated with the anti-CTV-RdRp serum, suggesting that the 50-kDa and 10-kDa proteins were fragments of the 60-kDa CTV-RdRp fusion protein. When the expression of the RdRp was analyzed at different times during in vitro translation, the 60-kDa and 50-kDa proteins were detected at all time points, and a small amount of the 10-kDa protein was detected after 30 min of translation. These results suggest that the CTV-RdRp may also be cleaved in vitro in the rabbit reticulocyte lysate.

Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus.

ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP-MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a 'double tag' construct, the GST-PCP-MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST-PCP and MT fragments. MT-His(6) was purified on Ni-NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly(588)/Gly(589) bond cleavage. The GST-PCP fragment purified on glutathione S-agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.

A replication-competent chimera of plant and animal viruses.

Human, animal, fungal, and plant viruses encode papain-like proteinases that function in polyprotein processing, RNA synthesis, and virus-host interactions. To compare the functional profiles of diverse papain-like proteinases, we replaced a proteinase gene of the beet yellows virus (BYV) with those derived from equine arteritis virus (EAV), foot-and-mouth disease virus (FMDV), and the fungal virus CHV1. We found that, although each of the foreign proteinases efficiently processed the viral polyprotein, only the EAV proteinase supported vigorous replication of the chimeric BYV in plant protoplasts. This result demonstrated that the proteinases of BYV and EAV, but not FMDV or CHV1, provide a function that is critical for genome replication and that is separable from polyprotein processing. Further characterization of the BYV-EAV chimera revealed that BYV proteinase is also required for virus invasion and cell-to-cell movement. Thus, the same viral protein can combine both replication-related functions shared by plant and animal viruses and specialized functions in virus-host interactions.

Ultrastructural localization and epitope mapping of the methyltransferase-like and helicase-like proteins of Beet yellows virus.

Monoclonal antibodies (MAbs) specific to the methyltransferase (MT) and helicase (HEL) domains of the closterovirus Beet yellows virus (BYV) were used for immunogold labelling of ultrathin sections of virus-infected Tetragonia expansa plants. MAbs 4A2 and 4A5 from the MT panel, and 1C4 from the HEL panel, specifically labelled distinct closterovirus-induced membranous structures, the 'BYV-type vesicles', thus suggesting that the closterovirus MT-like and HEL-like proteins co-localize in these structures. Probing of the MT and HEL MAbs with synthetic octapeptides spanning the sequences of the recombinant MT and HEL fragments that had been used as immunogens showed that 4A5 and 4A2 recognized a single epitope, SRLLENET (aa 686-692 in the BYV 1a protein), and 1C4 reacted with the DDPF epitope (aa 2493-2496). These epitopes apparently reside on the exposed parts of the membrane-associated molecules of the closterovirus MT-like and HEL-like proteins. Two other epitopes determined for the MT MAbs that were nonreactive in the immunogold labelling, namely TMVTPGEL (aa 750-757; MAbs 3C5, 4B4 and 4C5) and SREQLVEA (aa 806-813; MAb 2A4), are possibly buried in the MT domain fold or shielded by membranes or other proteins involved in the viral replicative complex.

Leader proteinase of the beet yellows closterovirus: mutation analysis of the function in genome amplification.

The beet yellows closterovirus leader proteinase (L-Pro) possesses a C-terminal proteinase domain and a nonproteolytic N-terminal domain. It was found that although L-Pro is not essential for basal-level replication, deletion of its N-terminal domain resulted in a 1, 000-fold reduction in RNA accumulation. Mutagenic analysis of the N-terminal domain revealed its structural flexibility except for the 54-codon-long, 5'-terminal element in the corresponding open reading frame that is critical for efficient RNA amplification at both RNA and protein levels.

Detection of beet yellows closterovirus methyltransferase-like and helicase-like proteins in vivo using monoclonal antibodies.

In the positive-stranded RNA genome of beet yellows closterovirus (BYV), the 5'-terminal ORF 1a encodes a 295 kDa polyprotein with the domains of papain-like cysteine proteinase, methyltransferase (MT) and helicase (HEL), whereas ORF 1b encodes an RNA-dependent RNA polymerase. Eleven and five hybridoma cell lines secreting monoclonal antibodies (MAbs) were derived from mice injected with the bacterially expressed fragments of the BYV 1a product encompassing the MT and HEL domains, respectively. On immunoblots of protein from BYV-infected Tetragonia expansa plants, four MAbs against the MT recognized a approximately 63 kDa protein, and two MAbs against the HEL recognized a approximately 100 kDa protein. Both the methyltransferase-like protein and the helicase-like protein were found mainly in the fractions of large organelles (P1) and membranes (P30) of the infected plants. These data clearly indicate that (i) the BYV methyltransferase-like and helicase-like proteins, like other related viral enzymes, are associated with membrane compartments in cells, and (ii) the 1a protein, apart from the cleavage by the leader papain-like proteinase that is expected to produce the 66 kDa and 229 kDa fragments, undergoes additional processing by a virus-encoded or cellular proteinase.

Nucleotide sequence and organization of ten open reading frames in the genome of grapevine leafroll-associated virus 1 and identification of three subgenomic RNAs.

The genome of Grapevine leafroll-associated virus 1 (GLRaV-1) was cloned and the sequence of 12394 nts determined. It contains 10 major open reading frames (ORFs) and a 3'-non-coding region lacking a poly(A) tract. The first ORF (ORF 1a) encodes a putative RNA helicase at the C-terminal portion of an apparently larger protein. The downstream ORF, 1b, overlaps ORF 1a and lacks an initiation codon. This ORF encodes an RNA-dependent RNA polymerase of M(r) 59276. ORF 2 encodes a small hydrophobic protein of M(r) 6736, and ORF 3 encodes a homologue of the HSP70 family of heat shock proteins and has an M(r) of 59500. ORF 4 encodes a protein with an M(r) of 54648 that shows similarity to the corresponding proteins of other closteroviruses. ORF 5 encodes the viral coat protein (CP) with an M(r) of 35416. The identity of this ORF as the CP gene was confirmed by expression in Escherichia coli and testing with the viral antibody. ORFs 6 and 7 code for two CP-related products with M(r) of 55805 and 50164, respectively. ORFs 8 and 9 encode proteins of M(r) 21558 and 23771 with unknown functions. Using DNA probes to different regions of the GLRaV-1 sequence, three major 3'-coterminal subgenomic RNA species were identified and mapped on the GLRaV-1 genome. Phylogenetic analyses of the individual genes of GLRaV-1 demonstrated a closer relationship between GLRaV-1 and GLRaV-3 than with other closteroviruses.