Replication of an incomplete alfalfa mosaic virus genome in plants transformed with viral replicase genes

The cell-to-cell transport of plant viruses depends on one or more virus-encoded movement proteins (MPs). Some MPs are integral membrane proteins that interact with the membrane of the endoplasmic reticulum, but a detailed understanding of the interaction between MPs and biological membranes has been lacking. The cell-to-cell movement of the Prunus necrotic ringspot virus (PNRSV) is facilitated by a single MP of the 30K superfamily. Here, using a myriad of biochemical and biophysical approaches, we show that the PNRSV MP contains only one hydrophobic region (HR) that interacts with the membrane interface, as opposed to being a transmembrane protein. We also show that a proline residue located in the middle of the HR constrains the structural conformation of this region at the membrane interface, and its replacement precludes virus movement.Plant viruses encode movement proteins (MPs) that mediate the intra-and intercellular spread of the viral genome via plasmodesmata, membranous channels that traverse the walls of plant cells and enable intercellular transport and communication. There is a range of diversity in the number and type of viral proteins required for viral movement (21). Research on tobacco mosaic virus (TMV) has played a leading role in understanding MP activity (2). The genome of TMV encodes a single 30-kDa multidomain protein, the namesake of the 30K superfamily (7). Viral RNA is associated with the membrane of the endoplasmic reticulum (ER) and microtubules in the presence of this MP (23,30).A large number of plant viruses have 30K MPs, which share common abilities, including binding nucleic acids, localizing and increasing the size exclusion limit of plasmodesmata, and interacting with the ER membrane. A topological model has been proposed in which the TMV MP has two putative transmembrane (TM) helices, both the N and C termini oriented toward the cytoplasm, and a short loop exposed in the ER lumen (4). There is less experimental information for other 30K MPs, but they are likely to have some membrane interaction.Direct experimental evidence of the integration of MPs into the membrane has been obtained only for small hydrophobic MPs that do not belong to the 30K superfamily. There are two TM segments in the p9 protein of carnation mottle virus (41), whereas the p6 protein of beet yellow virus (29) and the p7B protein of melon necrotic spot virus (22) have a single TM segment. In viruses with genomes that include three partially overlapping open reading frames, termed the triple-gene block (TGB), all three TGB proteins are required for movement where the two smaller proteins, TGBp2 and TGBp3, are also TM proteins (24). Furthermore, cross-linking experiments with carnation mottle virus p9 protein demonstrated that its membrane insertion occurs cotranslationally in a signal recognition particle-dependent manner and throughout the cellular membrane integration components, the translocon (33, 34).Prunus necrotic ringspot virus (PNRSV) is a tripartite, positive-strand RNA virus in the genus Ilarvirus ...

Section: Methodsmentioning

confidence: 99%

Plant Virus Cell-to-Cell Movement Is Not Dependent on the Transmembrane Disposition of Its Movement Protein

Martínez-Gil

Sánchez‐Navarro

Cruz

et al. 2009

“…As a positive control, leaves were infiltrated with a mixture of bacteria containing the wt constructs pMOGR12 and pMOGR3. Total RNA was isolated from the leaves 5 days after infiltration and analyzed by Northern blot hybridization as described previously (31 To analyze if virus containing the RNA 1 chimeras was fully infectious, RNA was extracted from the infiltrated leaves and inoculated onto N. benthamiana plants as described previously (21). Total RNA was isolated from the leaves 5 days after inoculation and analyzed by Northern blot hybridization.…”

mentioning

confidence: 99%

The 5′ Untranslated Region of Alfalfa Mosaic Virus RNA 1 Is Involved in Negative-Strand RNA Synthesis

Vlot

Bol

2003

Self Cite

The three genomic RNAs of alfalfa mosaic virus each contain a unique 5 untranslated region (5 UTR). Replacement of the 5 UTR of RNA 1 by that of RNA 2 or 3 yielded infectious replicons. The sequence of a putative 5 stem-loop structure in RNA 1 was found to be required for negative-strand RNA synthesis. A similar putative 5 stem-loop structure is present in RNA 2 but not in RNA 3.Alfalfa mosaic virus (AMV) is a positive-strand tripartite RNA virus belonging to the family Bromoviridae (reviewed in reference 2). RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively. P1 contains a methyltransferase-like domain in its N-terminal half, as well as a helicase-like domain in its C-terminal half (6,18). Residues in P1 that are highly conserved among alphaviral methyltransferase proteins or superfamily I helicases are required for AMV RNA replication (29,30). P2 is the viral RNA-dependent RNA polymerase (RdRp) protein (16). RNA 3 encodes the movement protein P3, as well as the coat protein, CP, which is translated from subgenomic RNA 4. The three genomic RNAs of AMV are not infectious unless either CP or CP-mRNA 4 is present in the inoculum (2). It has been found that addition of a poly(A) tail to the 3Ј ends of the AMV RNAs partially replaces the requirement for CP in the inoculum (13). Moreover, binding of CP to the 3Ј untranslated regions (UTRs) of RNAs 3 and 4 strongly enhanced translation of these RNAs (13; L. Neeleman, H. J. M. Linthorst, and J. F. Bol, submitted for publication).AMV RNAs are flanked by 5Ј and 3Ј UTRs that are highly structured and presumed to contain cis-acting elements involved in translation and replication (14,15,22,23,24,25,27,28). The 3Ј UTRs of the three RNAs consist of a common 3Ј-terminal sequence of 145 nucleotides (nt) preceded by 18 to 34 nt that are unique for each RNA. It has been shown that RNA 3 with its 3Ј UTR replaced with that of either RNA 1 or 2 is replicated in P12 plants at the level of wild-type (wt) RNA 3 (25). P12 plants express P1 and P2 from nuclear transgenes and support replication of RNA 3 and synthesis of RNA 4 (21). In AMV strain 425, the 5Ј UTRs of RNAs 1 and 2 consist of 100 and 54 nt, respectively, while the 5Ј UTR of RNA 3 consists of 345 nt that are predicted to fold into a complex structure (24). The 5Ј UTR of RNA 3 contains several 27-nt repeats that encompass ICR 2-like motifs (24). These motifs are homologous to box B elements, which are present in brome mosaic virus (BMV) RNAs and mediate recruitment of the BMV RNAs to viral replication complexes (3,19,20,24). The 5Ј UTRs of AMV RNAs 1 and 2 do not contain recognizable ICR 2-like motifs. However, the 5Ј-terminal 11 nt of RNAs 1 and 2 are identical, and the 5Ј UTRs of both RNAs contain a predicted 5Ј-terminal stem-loop structure consisting of a 12-bp stem and a 4-nt loop (M fold) ( Fig. 1A and B). Interestingly, replication of RNA 3 with its 5Ј UTR replaced with that of either RNA 1 or 2 was not supported in P12 plants (27).Replication of AMV RNA 1 carrying the 5 UTR of either RNA 2 or 3. In the ...

“…Previously, it was shown that transgenic tobacco plants expressing the P1 protein (P1 plants) supported the replication of AMV RNAs 2 and 3 in the absence of RNA 1 (35). Inoculation of P1 plants or protoplasts with the three genomic RNAs resulted in coreplication of RNA 1 with RNAs 2 and 3.…”

Section: Resultsmentioning

confidence: 99%

“…Functions involved in replication of RNA 2. Transgenic tobacco plants expressing the P2 protein (P2 plants) support the replication of AMV RNAs 1 and 3 in the absence of RNA 2 (35). The transgenically expressed P2 did not interfere with the coreplication of wt RNA 2 with RNAs 1 and 3 but did not support coreplication of RNA 2 with a mutation changing the GDD sequence in the polymerase domain of the P2 protein into a GVD sequence (mutant GVD) (Fig.…”

Section: Vol 77 2003 Functions Of Amv Replicase Proteins 10795mentioning

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.