Alfalfa Mosaic Virus Replicase Proteins P1 and P2 Interact and Colocalize at the Vacuolar Membrane

The replication-associated proteins encoded by Cucumber mosaic virus (CMV), the 1a and 2a proteins, were detected by immunogold labeling in two host species of this virus, tobacco (Nicotiana tabacum) and cucumber (Cucumis sativus). In both hosts, the 1a and 2a proteins colocalized predominantly to the vacuolar membranes, the tonoplast. While plus-strand CMV RNAs were found distributed throughout the cytoplasm by in situ hybridization, minus-strand CMV RNAs were barely detectable but were found associated with the tonoplast. In both cucumber and tobacco, 2a protein was detected at higher densities than 1a protein. The 1a and 2a proteins also showed quantitative differences with regard to tissue distributions in tobacco and cucumber. About three times as much 2a protein was detected in CMV-infected cucumber tissues as in CMV-infected tobacco tissues. In tobacco, high densities of these proteins were observed only in vascular bundle cells of minor veins. In contrast, in cucumber, high densities of 1a and 2a proteins were observed in mesophyll cells, followed by epidermis cells, with only low levels being observed in vascular bundle cells. Differences were also observed in the distributions of 2a protein and capsid protein in vascular bundle cells of the two host species. These observations may represent differences in the relative rates of tissue infection in different hosts or differences in the extent of virus replication in vascular tissues of different hosts.

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

In Situ Localization and Tissue Distribution of the Replication-Associated Proteins of Cucumber Mosaic Virus in Tobacco and Cucumber

Cillo¹,

Roberts²,

Palukaitis³

2002

“…3B, lanes 13 and 14). We note, however, that the CP/ RNA ratios are different between the two assays and that the viral polymerase preparation is naturally contaminated with CP (22,26), which would result in even higher CP/RNA ratios. Nevertheless, comparison of pk1/hp2 with pk3/hp2 shows that a stronger pseudoknot is capable of preventing CP of inhibiting minus-strand synthesis at a CP/RNA ratio of 0.5 to 1.…”

Section: Resultsmentioning

confidence: 98%

In Vitro and In Vivo Studies of the RNA Conformational Switch in Alfalfa Mosaic Virus

Chen

Olsthoorn

2010

The 3 termini of Alfalfa mosaic virus (AMV) RNAs adopt two mutually exclusive conformations, a coat protein binding (CPB) and a tRNA-like (TL) conformer, which consist of a linear array of stem-loop structures and a pseudoknot structure, respectively. Previously, switching between CPB and TL conformers has been proposed as a mechanism to regulate the competing processes of translation and replication of the viral RNA (R. C. L. Olsthoorn et al., EMBO J. 18:4856-4864, 1999). In the present study, the switch between CPB and TL conformers was further investigated. First, we showed that recognition of the AMV 3 untranslated region (UTR) by a tRNA-specific enzyme (CCA-adding enzyme) in vitro is more efficient when the distribution is shifted toward the TL conformation. Second, the recognition of the 3 UTR by the viral replicase was similarly dependent on the ratio of CBP and TL conformers. Furthermore, the addition of CP, which is expected to shift the distribution toward the CPB conformer, inhibited recognition by the CCA-adding enzyme and the replicase. Finally, we monitored how the binding affinity to CP is affected by this conformational switch in the yeast three-hybrid system. Here, disruption of the pseudoknot enhanced the binding affinity to CP by shifting the balance in favor of the CPB conformer, whereas stabilizing the pseudoknot did the reverse. Together, the in vitro and in vivo data clearly demonstrate the existence of the conformational switch in the 3 UTR of AMV RNAs.Alfalfa mosaic virus (AMV) is a plant virus that belongs to one of the five genera in the family Bromoviridae, whose genomes consist of three genomic RNAs (RNAs 1, 2, and 3) and one subgenomic RNA (RNA4) that are capped at the 5Ј end and lack polyadenylation at the 3Ј terminus (3). RNAs 1 and 2 encode the viral subunits P1 and P2 of the replicase, respectively. RNA3 encodes the movement protein and serves as a template for the synthesis of RNA4, which encodes the coat protein (CP).The role of AMV CP has been the subject of extensive research in the past four decades. Initially, it was found that, in contrast to RNAs of the Bromo-, Cucumo-, and Oleavirus genera, the genomic RNAs of AMV and the closely related genus Ilarvirus were not infectious as such but required the presence of CP in the inoculum (15). This phenomenon was called genome activation and was long considered to compensate for the lack of a tRNA-like structure (TLS) at the 3Ј end of their genomic RNAs, a prominent feature of bromo-and cucumovirus RNAs (3). However, in 1999 we demonstrated that the 3Ј end of AMV RNAs can adopt an alternative conformation that shows many structural similarities to the TLS of other Bromoviridae, although it could not be charged with an amino acid (20). The tRNA-like (TL) conformation (Fig. 1) turned out to be the replicative form of the 3Ј termini (19,20), whereas the other, coat protein binding (CPB), conformer was subsequently shown to be required for translation (16)(17)(18). Although other models have been forwarded (9), we have proposed that ...

“…Samples from the P30 pellets corresponding to 50 mg of infiltrated tissue were loaded onto a Western blot. P1 and P2 were detected using antisera to the C-terminal amino acids 1100 to 1120 of P1 and the N terminus of P2 (36,38). Western blotting was performed with Hybond-P polyvinylidene difluoride membranes (Amersham Pharmacia Biotech).…”

Section: Vol 77 2003 Functions Of Amv Replicase Proteins 10791mentioning

confidence: 99%

Coordinate Replication of Alfalfa Mosaic Virus RNAs 1 and 2 Involves cis - and trans -Acting Functions of the Encoded Helicase-Like and Polymerase-Like Domains

Vlot

Laros

Bol

2003

Self Cite

RNAs 1 and 2 of the tripartite genome of alfalfa mosaic virus encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and coat protein. Transient expression of wild-type (wt) and mutant viral RNAs and proteins by agroinfiltration of plant leaves was used to study cis-and trans-acting functions of the helicase-like domain in P1 and the polymerase-like domain in P2. Three mutations in conserved motifs of the helicase-like domain of P1 affected one or more steps leading to synthesis of minus-strand RNAs 1, 2, and 3. In leaves containing transiently expressed P1 and P2, replication of wt but not mutant RNA 1 was observed. Apparently, the transiently expressed P1 could not complement the defect in replication of the RNA 1 mutant. Moreover, the transiently expressed wt replicase supported replication of RNA 2, but this replication was blocked in trans by coexpression of mutant RNA 1. However, expression of mutant RNA 1 did not interfere with the replication of RNA 3 by the wt replicase. Similarly, a mutation in the GDD motif encoded by RNA 2 could not be complemented in trans and affected the replication of RNA 1 by a wt replicase, while replication of RNA 3 remained unaffected. In competition assays, the transient wt replicase preferentially replicated RNA 3 over RNAs 1 and 2. The results indicate that one or more functions of P1 and P2 act in cis and point to the existence of a mechanism that coordinates the replication of RNAs 1 and 2.