Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA.

On entering a host cell, positive-strand RNA virus genomes have to serve as messenger for the translation of viral proteins. Efficient translation of cellular messengers requires interactions between initiation factors bound to the 5'-cap structure and the poly(A) binding protein bound to the 3'-poly(A) tail. Initiation of infection with the tripartite RNA genomes of alfalfa mosaic virus (AMV) and viruses from the genus Ilarvirus requires binding of a few molecules of coat protein (CP) to the 3' end of the nonpolyadenylated viral RNAs. Moreover, infection with the genomic RNAs can be initiated by addition of the subgenomic messenger for CP, RNA 4. We report here that extension of the AMV RNAs with a poly(A) tail of 40 to 80 A-residues permitted initiation of infection independently of CP or RNA 4 in the inoculum. Specifically, polyadenylation of RNA 1 relieved an apparent bottleneck in the translation of the viral RNAs. Translation of RNA 4 in plant protoplasts was autocatalytically stimulated by its encoded CP. Mutations that interfered with CP binding to the 3' end of viral RNAs reduced translation of RNA 4 to undetectable levels. Possibly, CP of AMV and ilarviruses stimulates translation of viral RNAs by acting as a functional analogue of poly(A) binding protein or other cellular proteins.

Role of the 3'-untranslated regions of alfalfa mosaic virus RNAs in the formation of a transiently expressed replicase in plants and in the assembly of virions.

Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of Nicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3'-untranslated regions (UTRs) of both RNAs deleted (R1Delta/2Delta construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1Delta/2Delta construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1Delta/2 (3'-UTR of RNA 1 deleted), R1/2Delta (3'-UTR of RNA 2 deleted), or R1Delta/2Delta plus R3. This demonstrates that the 3'-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3'-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3'-termini of AMV RNAs is not required for assembly of virus particles.

Cis-acting functions of alfalfa mosaic virus proteins involved in replication and encapsidation of viral RNA.

cDNA clones of RNAs 1 and 2 of alfalfa mosaic virus (AMV) were slightly modified to permit transcription of infectious RNAs with T7 RNA polymerase. Together with transcripts of an available clone of AMV RNA 3, these transcripts were used to study cis- and trans-acting functions of AMV proteins in protoplasts from nontransgenic tobacco plants and from plants transformed with the P1 and P2 genes, encoded by RNAs 1 and 2, respectively. Transgenic P1 was unable to complement mutations in the P1 gene in RNA 1, pointing to a cis-acting function of P1 in RNA 1 replication. A study of the replication of RNA 3 mutants in nontransgenic protoplasts revealed that coat protein (CP) expressed from RNA 3 in the inoculum is required in trans for replication and encapsidation of RNAs 1 and 2 but is required in cis for replication and encapsidation of RNA 3. CP is required in the inoculum to initiate infection of nontransgenic plants and protoplasts. When protoplasts expressing both P1 and P2 (P12 protoplasts) were infected with RNAs 1, 2, and 3, initiation of replication of RNAs 1 and 2 required the presence of CP in the inoculum, whereas the initiation of replication of RNA 3 did not. This demonstrated that CP expressed from RNA 3 cannot substitute for the early function of CP in the inoculum. The results showed that CP in the inoculum is required to permit viral minus-strand RNA synthesis, whereas CP expressed from RNA 3 after the initiation of infection is required for plus-strand RNA synthesis.

Mutations in coat protein binding sites of alfalfa mosaic virus RNA 3 affect subgenomic RNA 4 accumulation and encapsidation of viral RNAs.

The 3'-untranslated regions (3'-UTRs) of the three RNAs of alfalfa mosaic virus (AMV) contain a specific binding site for coat protein (CP) and act as a promoter for minus-strand RNA synthesis by the purified AMV RNA-dependent RNA polymerase (RdRp) in an in vitro assay. Binding of CP to the viral RNAs is required to initiate infection. The sequence of the 3'-terminal 39 nucleotides of AMV RNA 3 can be folded into two stem-loop structures flanked by three single-stranded AUGC sequences and represents a CP binding site. Mutations in this sequence that are known to interfere with CP binding in vitro were introduced into an infectious clone of RNA 3, and mutant RNA transcripts were used as templates in the in vitro RdRp assay and to infect protoplasts and plants. Mutation of AUGC motif 2 or disruption of the stem of the 3'-proximal hairpin 1 interfered with CP binding in vitro but not with minus-strand promoter activity in vitro or replication of RNA 3 in vivo. However, hairpin 1 appeared to be essential for encapsidation of RNA 3. Reversion of three G-C base pairs in hairpin 1 had no effect on CP binding but interfered with minus-strand promoter activity in vitro and with RNA 3 replication in vivo. It is concluded that the viral RdRp and CP recognize different elements in the 3'-UTRs of AMV RNAs. Moreover, several mutations that interfered with CP binding in vitro interfered with the accumulation in vivo of RNA 4, the subgenomic messenger for CP, but not with the accumulation of RNA 3.

Comparison of the role of 5' terminal sequences of alfalfa mosaic virus RNAs 1, 2, and 3 in viral RNA replication.

The 5' untranslated regions (UTRs) of the genomic RNAs 1, 2, and 3 of alfalfa mosaic virus (AMV) are 100, 54, and 345 nucleotides (nt) long, respectively, and lack extensive sequence similarity to each other. RNA 3 encodes the movement protein P3 and the coat protein and can be replicated in transgenic tobacco plants expressing the replicase proteins P1 and P2 (P12 plants). 5' Cis-acting sequences involved in RNA 3 replication have been shown to be confined to the 5' UTR. When the 5' UTR of RNA 3 was replaced by the 5' UTRs of RNAs 1 or 2, the recombinant RNA was not infectious to P12 plants. Also, when the P3 gene in RNA 3 was put under the control of a subgenomic promoter and the 5' UTR of this RNA was replaced by 5' terminal RNA 1 sequences of 103 to 860 nt long or RNA 2 sequences of 57 to 612 nt long, no accumulation of the hybrid RNAs was observed. Deletion of the 5' 22 nucleotides of RNA 3 resulted in the accumulation of a major progeny that lacked the 5' 79 nt. However, when the 5' 22 nucleotides of RNA 3 were replaced by the complete 5' UTR of RNA 1 or 5' sequences of RNAs 1, 2, or 3 with a length of 5 to 15 nt, accumulation of the full-length mutant RNAs was observed. The effect of mutations in the 5' viral sequences of 5 to 15 nt was analyzed. It is concluded that although elements within nucleotides 80-345 of the 5' UTR of RNA 3 are sufficient for replication, a specific sequence of 3 to 5 nt is required to target the replicase to an initiation site corresponding to the 5' end of the RNA.

Functional equivalence of common and unique sequences in the 3' untranslated regions of alfalfa mosaic virus RNAs 1, 2, and 3.

The 3' untranslated regions (UTRs) of alfalfa mosaic virus (AMV) RNAs 1, 2, and 3 consist of a common 3'-terminal sequence of 145 nucleotides (nt) and upstream sequences of 18 to 34 nt that are unique for each RNA. The common sequence can be folded into five stem-loop structures, A to E, despite the occurrence of 22 nt differences between the three RNAs in this region. Exchange of the common sequences or full-length UTRs between the three genomic RNAs did not affect the replication of these RNAs in vivo, indicating that the UTRs are functionally equivalent. Mutations that disturbed base pairing in the stem of hairpin E reduced or abolished RNA replication, whereas compensating mutations restored RNA replication. In vitro, the 3' UTRs of the three RNAs were recognized with similar efficiencies by the AMV RNA-dependent RNA polymerase (RdRp). A deletion analysis of template RNAs indicated that a 3'-terminal sequence of 127 nt in each of the three AMV RNAs was not sufficient for recognition by the RdRp. Previously, it has been shown that this 127-nt sequence is sufficient for coat protein binding. Apparently, sequences required for recognition of AMV RNAs by the RdRp are longer than sequences required for CP binding.

The 5' terminal sequence of alfalfa mosaic virus RNA 3 is dispensable for replication and contains a determinant for symptom formation.

Transgenic P12 tobacco plants, transformed with the replicase genes P1 and P2 of alfalfa mosaic virus (AIMV), can be infected with RNA 3 of the tripartitite AIMV genome or with a DNA copy of RNA 3 fused to the CaMV 35S promoter and nos terminator. The effect of various modifications on the infectivity of the 35S/cDNA 3 construct to P12 plants was studied. When nonviral sequences ranging from 11 to 200 bp were inserted between the 35S promoter and cDNA 3, the infection became dependent on addition of coat protein (CP) to the inoculum. About 80% of the progeny RNAs resulting from these infections were full-length and had lost the nonviral sequence, whereas 20% were truncated by a deletion of the 5' terminal 79 nucleotides (nt). When the sequence corresponding to the 5' terminal 22 nt of RNA 3 was deleted from the 35S/cDNA 3 construct, the clone was as infectious as the wild type (wt), provided that CP was added to the inoculum, but only progeny RNA with a 5' terminal deletion of 79 nt was produced. The 5' truncated RNA 3 molecules induced necrotic ringspot-like symptoms on P12 tobacco plants, whereas wt RNA 3 did not induce detectable symptoms on these plants. It is proposed that in the infections with the modified 35S/cDNA 3 clones, CP is required in the inoculum to permit internal initiation of plus-strand RNA 3 synthesis on 3'-extended or 3'-truncated minus-strand RNA templates. Evidence was obtained that minus-strand RNA 3 synthesized under the control of the 35S promoter was not infectious to P12 plants.

Ability of tobacco streak virus coat protein to substitute for late functions of alfalfa mosaic virus coat protein.

The coat protein (CP) of tobacco streak virus (TSV) can substitute for the early function of alfalfa mosaic virus (AIMV) CP in genome activation. Replacement of the CP gene in AIMV RNA 3 with the TSV CP gene and analysis of the replication of the chimeric RNA indicated that the TSV CP could not substitute for the function of AIMV CP in asymmetric plus-strand RNA accumulation but could encapsidate the chimeric RNA and permitted a low level of cell-to-cell transport.

Early and late functions of alfalfa mosaic virus coat protein can be mutated separately.

To investigate the role of alfalfa mosaic virus coat protein (CP) in genome activation, asymmetric plus-strand RNA accumulation, and cell-to-cell spread of the virus, mutations were made in the CP gene and putative CP binding sites in the 3'-untranslated region (UTR) of RNA 3. Mutants that produced no CP-related peptide or CP with an N-terminal deletion of 20 amino acids were defective in all three functions. Insertion of several nonviral amino acids at position 85 of CP had little effect on genome activation and plus-strand RNA accumulation but abolished cell-to-cell spread. A mutant encoding CP with a C-terminal deletion of 21 amino acids was defective in plus-strand RNA accumulation but showed substantial levels of genome activation and cell-to-cell spread. Mutations in the 3'-UTR that interfered with CP binding affected plus-strand RNA accumulation and cell-to-cell spread. Neither CP nor CP binding sites at the 3'-end of RNA 3 were required for minus-strand RNA accumulation. The results demonstrate that early and late functions of CP can be mutated separately, indicating that different domains of CP are involved in the three functions investigated.

The 3'-untranslated region of alfalfa mosaic virus RNA 3 contains at least two independent binding sites for viral coat protein.

The 3'-termini of the three genomic RNAs of alfalfa mosaic virus contain a common sequence of 145 nucleotides (nt) with a specific binding site for coat protein (CP). This sequence consists of several stem/loop structures interspersed with single-stranded AUGC-motifs; in RNA 3 this folding pattern is extended to a region upstream of the homologous sequence. By band-shift assays a minimum of two specific binding sites for CP were identified near the 3'-end of RNA 3. Site 1 consists of the region between nt 11 and 127 from the 3'-end and contains two AUGC-motifs. Site 2 is located between nt 133 and 208 from the 3'-end in a sequence that is largely unique to RNA 3 and contains also two AUGC-motifs. Deletion studies revealed that the two sites could bind CP independently of each other and permitted the identification of sequence elements that are essential for the activity of each site. By site-directed mutagenesis it was shown that the AUGC-motifs are important for binding of CP to both sites. These binding sites may play a role in the phenomenon that each genomic RNA has to be complexed with a few CP molecules to initiate infection. Later in the replication cycle they may act as origins for the assembly of virus particles.

Infection of tobacco with alfalfa mosaic virus cDNAs sheds light on the early function of the coat protein.

Coat protein (CP) is required in the inoculum to initiate infection of plants with the three genomic RNAs of alfalfa mosaic virus. Inoculation of plants with DNA copies of RNAs 1, 2, and 3, fused to the 35S promoter, resulted in virus replication but the infection level was increased several-fold by addition of CP to the inoculum. When one of the three cDNAs was replaced by its corresponding RNA molecule there was no infection unless CP was present in the inoculum. Plants transformed with a DNA copy of RNA 1 or RNA 2 could be infected with mixtures of cDNAs 2 and 3 or cDNAs 1 and 3, respectively. Again, when one cDNA in the inoculum was replaced by its corresponding RNA, infection depended on the presence of CP in the inoculum. However, when DNA copies of both RNA 1 and RNA 2 were present in the plant genome, the plants became infected after inoculation with cDNA 3 or RNA 3 without any requirement for CP. It is concluded that the necessity of CP in the inoculum depends on the timing of the production of the replicase subunits P1 and P2, encoded by RNAs 1 and 2, respectively, and on the availability of viral template RNAs once the replication complex has been formed. Models to explain the early function of CP are discussed.

Role of the 5' leader sequence of alfalfa mosaic virus RNA 3 in replication and translation of the viral RNA.

RNA 3 of alfalfa mosaic virus (AIMV) encodes the movement protein P3 and the viral coat protein which is translated from the subgenomic RNA 4. The 5'-leader sequences of RNA 3 of AIMV strains S, A, and Y differ in length from 314 to 392 nucleotides and contain a variable number of internal control regions of type 2 (ICR2 motifs) each located in a 27 nt repeat. Infectious cDNA clones were used to exchange the leader sequences of the three strains. This revealed that the leader sequence controls the specific ratio in which RNAs 3 and 4 are synthesized for each strain. In addition, it specifies strain specific differences in the kinetics of P3 accumulation in plants. Subsequent deletion analysis revealed that a 5'-sequence of 112 nt containing one ICR2 motif was sufficient for a 10 to 20% level of RNA 3 accumulation in protoplasts and a delayed accumulation in plants. An additional leader sequence of maximally 114 nt, containing two ICR2 motifs, was required to permit wildtype levels of RNA 3 accumulation. The effect of deletions in the leader sequence on P3 synthesis in vitro and in vivo was investigated.

Role of alfalfa mosaic virus coat protein in regulation of the balance between viral plus and minus strand RNA synthesis.

Replication of wild type RNA 3 of alfalfa mosaic virus (AIMV) and mutants with frameshifts in the P3 or coat protein (CP) genes was studied in protoplasts from tobacco plants transformed with DNA copies of AIMV RNAs 1 and 2. Accumulation of viral plus and minus strand RNAs was monitored with strand-specific probes. A frameshift in the P3 gene did not change the asymmetry in plus/minus strand accumulation observed for the wild type. A frameshift early in the CP gene resulted in a 100-fold reduction in plus strand accumulation and a 3- to 10-fold increase in minus strand accumulation. A frameshift late in the CP gene caused a similar reduction in plus strand accumulation but had no effect on minus strand accumulation. This latter mutant accumulated nearly wild type levels of a truncated CP molecule. Apparently, wild type AIMV CP up-regulates plus strand accumulation and down-regulates minus strand accumulation and these two functions can be mutated separately.

Deletion analysis of cis- and trans-acting elements involved in replication of alfalfa mosaic virus RNA 3 in vivo.

DNA copies of alfalfa mosaic virus (AIMV) RNA 3 were transcribed in vitro into RNA molecules with deletions in coding and noncoding sequences. The replication of these transcripts was studied in protoplasts from transgenic tobacco plants expressing DNA copies of AIMV RNAs 1 and 2. Deletions in the 5'-proximal P3 gene, encoding the putative viral transport function, did not affect replication whereas deletions in the 3'-proximal coat protein gene reduced replication of RNA 3 by about 100-fold. Sequences required for the synthesis in protoplasts of RNA 4, the coat protein messenger, were more extensive than the subgenomic promoter characterized previously in an in vitro replicase assay. At the 5'-end of RNA 3 a sequence of 169 nucleotides was sufficient for replication whereas a sequence of 112 nucleotides was not. 3'-Terminal deletions up to 133 nucleotides reduced replication to a low but significant level. Further 3'-deletions abolished replication.

Complementation and recombination between alfalfa mosaic virus RNA3 mutants in tobacco plants.

Deletions were made in an infectious cDNA clone of alfalfa mosaic virus (AIMV) RNA3 and the replication of RNA transcripts of these cDNAs was studied in tobacco plants transformed with AIMV replicase genes (P12 plants). Previously, we found that deletions in the P3 gene did not affect accumulation of RNA3 in P12 protoplasts whereas deletions in the coat protein (CP) gene reduced accumulation 100-fold (A. C. van der Kuyl, L. Neeleman, and J. F. Bol, 1991, Virology 183, 687-694). In P12 plants deletions in the P3 gene reduced accumulation by about 200-fold and accumulation of CP deletion mutants was not detectable. When P12 plants were inoculated with a mixture of P3- and CP-deletion mutants, both mutants replicated efficiently and various amounts of full-length RNA3 molecules were formed by recombination. The observation that some P3 and CP mutants did not recombine at a detectable level after several passages in P12 plants demonstrated that mutations in the AIMV P3 and CP genes can be complemented in trans.

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

RNAs 1 and 2 of alfalfa mosaic virus (AIMV) encode proteins P1 and P2, respectively, both of which have a putative role in viral RNA replication. Tobacco plants were transformed with DNA copies of RNA1 (P1-plants), RNA2 (P2-plants) or a combination of these two cDNAs (P12-plants). All transgenic plants were susceptible to infection with the complete AIMV genome (RNAs 1, 2, and 3). Inoculation with incomplete mixtures of AIMV RNAs showed that the P1-plants were able to replicate RNAs 2 and 3, that the P2-plants were able to replicate RNAs 1 and 3, and that the P12-plants were able to replicate RNA3. Initiation of infection of nontransgenic plants, P1-plants, or P2-plants requires the presence of AIMV coat protein in the inoculum, but no coat protein was required to initiate infection of P12-plants with RNA3. Results obtained with P12-protoplasts supported the conclusion that coat protein plays an essential role in the replication cycle of AIMV RNAs 1 and 2.

Role of alfalfa mosaic virus coat protein gene in symptom formation.

On Samsun NN tobacco plants strains 425 and YSMV of alfalfa mosaic virus (AIMV) cause mild chlorosis and local necrotic lesions, respectively. DNA copies of RNA3 of both strains were transcribed in vitro into infectious RNA molecules. When the 425 and YSMV transcripts were inoculated to tobacco plants transformed with DNA copies of AIMV RNAs 1 and 2, they induced symptoms indistinguishable from those of the corresponding parent strains. Exchange of restriction fragments between the infectious clones showed that symptom expression was determined by the coat protein gene in RNA3. The sequence of YSMV RNA3 was determined and compared with the known sequence of 425 RNA3. When the codon for Gln-29 in the coat protein of strain 425 was mutated into the Arg codon present at this position in strain YSMV, the symptoms induced by the transcript on inoculated leaves changed from chlorosis to necrosis. Genetic determinants for the systemic response were more complex.

Biologically active transcripts of cloned DNA of the coat protein messenger of two plant viruses.

To initiate infection, a mixture of the three genomic RNAs of alfalfa mosaic virus (AIMV) has to be supplemented with a small amount of coat protein or RNA 4, the subgenomic messenger for coat protein. The possibility to replace RNA 4 in the inoculum by in vitro synthesized transcripts of a cloned DNA copy of the coat protein cistron was investigated using the SP6 transcription system. Transcripts with or without the cap structure m(7)G(5')ppp(5')G were both translated in vitro in viral coat protein, but only capped transcripts yielded an infectious mixture when added to the AIMV genomic RNAs. This indicates that the cap structure is essential to the in vivo translatin of RNA 4. Similar results were obtained with RNAs transcribed in vitro from a DNA copy of the putative coat protein cistron of tobacco streak virus (TSV). re]19850822 rv]19851203 ac]19860114.

Artifacts are responsible for the translational activity of polyribosome preparations isolated from alfalfa mosaic virus-infected tobacco leaves.

We observed that polyribosome preparations isolated from alfalfa mosaic virus (A1MV)-infected tobacco leaves were contaminated with virion-derived material which could not be removed completely by sucrose gradient centrifugation or by magnesium ion precipitation. Upon incubation of polyribosome preparations with S 100 extracts from reticulocyte lysates, viral-encoded proteins were produced. Aurintricarboxylic acid (ATA), an inhibitor of initiation of translation, was used to inhibit de novo translation of the RNAS contaminating the polyribosome preparations. ATA concentrations, which did not inhibit peptide chain elongation on in vitro-produced polyribosomes, completely inhibited the translational activity of the tobacco polyribosomes. Hence the protein synthetic capacity of the tobacco polyribosome preparations is due to de novo translation of virion-derived material by vacant ribosomes present in the complementing S 100 extract. Efforts to activate the tobacco polyribosomes remain unsuccessful.

Ribosomes are stalled during in vitro translation of alfalfa mosaic virus RNA 1.

In the presence of plant tRNAs the full-length translation product of alfalfa mosaic virus RNA 1 is produced in rabbit reticulocytes only at low mRNA concentration. At higher mRNA concentration translation is restricted to the 5' half of RNA 1. At high mRNA concentration the full-length product can be formed when additional plant tRNA and glutamine are supplied to the translation mixture. In contrast, in the presence of yeast or calf liver tRNA the translation pattern of alfalfa mosaic virus RNA 1 always results in the synthesis of the full-length product. Pulse-chase experiments in the presence of plant tRNAs show that the ribosomes pause at several positions in the 5' half of RNA 1. The pausing time is different at the different 'halting places'. Protein synthesis is resumed upon addition of glutamine, even when the addition is delayed for more than 3 h after the start of protein synthesis. Only one tRNA species, purified from wheat germ or tobacco, could promote full-length translation of RNA 1. This tRNA can be charged with glutamine. Analysis of the position of glutamine codons on RNA 1 shows a correlation between the positions of the CAA codons and the halting places of the ribosomes. The CAA codon (for any other codon) on its own cannot be responsible for the pausing of the ribosomes, since a variety of RNAs, known to contain all sense codons, are translated efficiently in rabbit reticulocyte lysates in the presence of plant tRNAs. Apparently other elements can restrict decoding of normal codons during protein chain elongation.