Time course of alfalfa mosaic virus RNA and coat protein synthesis in cowpea protoplasts

A study was made of the time course of the synthesis of viral plus-strand RNA, minus-strand RNA, and coat protein in alfalfa mosaic virus-infected Cowpea protoplasts. The three genomic RNAs were synthesized at different rates, as were their corresponding minus-strands. We conclude that viral RNA synthesis is regulated both at the level of minus-strand production and the level of plus-strand production. The synthesis of subgenomic RNA 4 was slower than that of its corresponding genomic RNA (RNA 3), indicating that an additional function, expressed later in infection, is required for production of subgenomic coat protein messenger. The data support a model for RNA 4 synthesis involving internal initiation by the RNA polymerase at the intercistronic junction in minus-strand RNA 3. The temporal relationship of the synthesis of RNA 3, RNA 4, and coat protein is discussed.     

West Nile virus genome cyclization and RNA replication require two pairs of long-distance RNA interactions

West Nile virus (WNV) genome cyclization and replication require two pairs of long-distance RNA interactions. Besides the previously reported 5'CS/3'CSI (conserved sequence) interaction, a 5'UAR/3'UAR (upstream AUG region) interaction also contributes to genome cyclization and replication. WNVs containing mutant 5'UARs capable of forming the 5'/3' viral RNA interaction were replicative. In contrast, WNV containing a 5'UAR mutation that abolished the 5'/3' viral RNA interaction was non-replicative; however, the replication defect could be rescued by a single-nucleotide adaptation that restored the 5'/3' RNA interaction. The 5'UAR/3'UAR interaction is critical for RNA synthesis, but not for viral translation. Antisense oligomers targeting the 5'UAR/3'UAR interaction effectively inhibited WNV replication. Phylogenic analysis showed that the 3'UAR could alternate between pairing with the 5'UAR or with the 3' end of the flaviviral genome. Therefore, the 5'UAR/3'UAR pairing may release the 3' end of viral genome (as a template) during the initiation of minus-strand RNA synthesis.     

Altered balance of the synthesis of plus- and minus-strand RNAs induced by RNAs 1 and 2 of alfalfa mosaic virus in the absence of RNA 3

The synthesis of viral plus-strand and minus-strand RNAs in cowpea protoplasts inoculated with mixtures of alfalfa mosaic virus nucleoproteins (B, M, Tb, and Ta) was analyzed by the Northern blotting technique. A mixture of B, M, and Tb induced the synthesis of plus-strand RNAs 1, 2, 3, and 4 and three minus-strand RNAs corresponding to RNAs 1, 2, and 3, respectively. Compared to this complete infection, a mixture of B and M induced the synthesis of a reduced amount of plus-strand RNAs 1 and 2 and a greatly enhanced amount of minus-strand RNAs 1 and 2. No detectable viral RNA synthesis was induced by mixtures of B and Tb or M and Tb. It is concluded that expression of genomic RNAs 1 and 2 results in the formation of a replicase activity that produces roughly equal amounts of viral plus- and minus-strand RNAs and that an RNA 3-encoded product, possibly the coat protein, is responsible for a switch to an asymmetric production of viral plus-strand RNA. The observation that no minus-strand corresponding to the subgenomic RNA 4 is produced suggests that recognition of the genome segments by the viral replicase involves sequences outside the 3'-terminal regions that are homologous to RNA 4.     

Comparative investigations on the coat protein binding sites of the genomic RNAs of alfalfa mosaic and tobacco streak viruses

The genomic RNAs of alfalfa mosaic virus (AIMV) and tobacco streak virus (TSV) form complexes with viral coat protein. These complexes were subjected to digestion with ribonuclease T1 and filtered onto Millipore filters. It was shown that the major coat protein binding sites are located at the 3' ends of the genomic RNA species of AIMV and TSV in both heterologous and homologous RNA-coat protein combinations. Internal coat protein binding sites were found as well. Although there is homology between the 3'-terminal sequences, no structural features could be observed that are common to all coat protein binding sites. The fact that TSV and AIMV coat protein can mutually activate each others genome combined with the fact that the major target site of both coat protein preparations is located at the 3' ends of the genomic RNAs favors the assumption that binding of the coat protein to the 3' ends is an initiation event of the replication cycle.     

A 5' RNA element promotes dengue virus RNA synthesis on a circular genome

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA. We demonstrated that RNA-RNA interactions between 5' and 3' end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5' end of the genome and reaches the site of initiation at the 3' end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.     

Alfalfa mosaic virus temperature-sensitive mutants. I. Mutants defective in viral RNA and protein synthesis

The replication in cowpea protoplasts of temperature-sensitive (ts) mutants of alfalfa mosaic virus (AIMV) was studied at the permissive (25 degrees) and the restrictive (30 degrees) temperature. Using the Northern blot hybridization technique, it was shown that at the restrictive temperature two RNA 1 mutants, Bts 03 and Bts 04, and two RNA 2 mutants, Mts 03 and Mts 04, were all defective in the synthesis of viral minus-strand RNA, whereas the synthesis of the plus-strand genomic RNAs 1, 2, and 3 and the subgenomic coat protein messenger, RNA 4, was relatively unimpaired. In Bts 04 inoculated protoplasts the RNA 4 produced at 30 degrees was translated into coat protein and viral RNA was encapsidated to give infectious virus. RNA 4 in Bts 03 and Mts 04 infected protoplasts was not translated into coat protein at 30 and consequently there was no assembly of infectious virus. Protein synthesis by Mts 03 was not investigated. A1MV RNAs 1 and 2 encoded proteins are both involved in the synthesis of viral minus-strand RNA and the translation of RNA 4 and possibly other viral messengers. The results with Bts 03 and Bts 04 show that the two functions of the RNA 1 encoded protein can be mutated separately.     

Alfalfa mosaic virus temperature-sensitive mutants. II. Early functions encoded by RNAs 1 and 2

Mutants Bts 03 and Mts 04 of alfalfa mosaic virus (AIMV) have temperature-sensitive mutations in genomic RNAs 1 and 2, respectively. These mutants are defective in the production of viral minus-strand RNA, coat protein, and infectious virus when assayed in cowpea protoplasts at the nonpermissive temperature (30 degrees). To determine the temperature-sensitive step in the replication cycle, mutant-infected protoplasts were shifted from an incubation temperature of 25 degrees (permissive temperature) to 30 degrees at different times during a 24-hr incubation period. For both mutants an initial incubation of infected protoplasts for 6 hr at 25 degrees was sufficient to permit a normal minus-strand RNA synthesis, translation of RNA 4 into coat protein, and assembly of infectious virus during the subsequent incubation at the nonpermissive temperature. Probably, AIMV RNAs 1 and 2 encoded proteins are produced early in infection and the mutant proteins are protected from inactivation at 30 degrees once they are incorporated in a functional structure.     

Activation of the alfalfa mosaic virus genome by viral coat protein in non-transgenic plants and protoplasts. The protection model biochemically tested

Summary.  In non-transgenic host plants and protoplasts alfalfa mosaic virus displays a strong need for coat protein when starting an infection cycle. The “protection model” states that the three viral RNAs must have a few coat protein subunits at their 3′ termini in order to protect them in the host cell against degradation by 3′- to- 5′ exoribonucleases [Neeleman L, Van der Vossen EAG, Bol JF (1993) Virology 196: 883–887]. We demonstrated that the naked genome RNAs are slightly infectious, if the inoculation is done at very high concentrations, or if it is preceded by an additional inoculation with the RNAs 1 and 2 (encoding subunits for the viral RNA polymerase). This could mean that the necessity for protection by coat protein is lost if the RNAs in large quantities can overcome the activity of the degrading enzymes, or are protected by association with the RNA polymerase, respectively. However, after having tested in protoplasts the survival of separately preinoculated naked RNA 1 during several hours before RNA 2 was inoculated, on the one hand, or of simultaneously inoculated RNAs 1 and 2, with cycloheximide in the medium during the first hours after inoculation, on the other hand, we had to conclude that the viral genome RNAs are quite stable in the cell in the absence of coat protein or RNA polymerase, respectively. This invalidates the protection model. Accommodation of the above findings by our published “messenger release model” for genome activation [Houwing CJ, Jaspars EMJ (1993) Biochimie 75: 617–621] is discussed. Received April 29, 1999/Accepted August 30, 1999     

Molecular cloning of clover yellow mosaic virus RNA: identification of coat protein coding sequences in vivo and in vitro

We have prepared double-stranded cDNA from clover yellow mosaic virus (CYMV) RNA and have created a library of cloned CYMV cDNAs in plasmid vectors. Cloned fragments ranging from 0.2 to 2.0 kbp have been mapped relative to the CYMV genome and to each other by digestion with restriction enzymes and by colony hybridization. Together, these cloned DNAs constitute almost 90% of the CYMV genome. Two overlapping plasmids whose inserts originate from the 3' portion of CYMV RNA arrest the synthesis of CYMV coat protein in vitro. A 0.44-kbp PstI fragment from one of these anneals to three CYMV-specific RNAs comprising the genomic RNA and two subgenomic RNAs of 2.1 and 1.0 kb isolated from polyribosomes from CYMV-infected broad bean leaves. RNA was enriched in the 1.0-kb subgenomic species by fractionating RNA extracted from purified preparations of CYMV or from polyribosomes isolated from infected leaves. Such RNA fractions directed the synthesis of CYMV coat protein in vitro indicating that the 1.0-kb subgenomic RNA is likely to be the coat protein messenger in vivo.     

The 3' end of hepatitis E virus (HEV) genome binds specifically to the viral RNA-dependent RNA polymerase (RdRp)

Hepatitis E virus (HEV) is the major cause of acute epidemic and sporadic hepatitis in the developing world. It is a positive-strand RNA virus with a genome length of about 7.2 kb. The replication mechanism of this virus is virtually unexplored. Identification of the regulatory elements involved in initiation of replication may help in designing specific inhibitors for therapy. In the positive-stranded RNA viruses the initiation of replication requires interaction of the 3' end of genome with its RNA-dependent RNA polymerase (RdRp) and possibly host-derived cofactors for synthesis of the minus-strand replicative intermediate. Secondary structure prediction of the conserved 3' end of the infectious HEV genome was carried out to identify possible stem-loop structures necessary for RNA-protein interaction and the model was confirmed by structure probing experiments. Electrophoretic mobility-shift assays showed specific binding of purified and refolded recombinant HEV RdRp protein to the 3' end of its RNA genome containing the poly(A) stretch. Mutations at the 3' end, in which the stem-loop structures were partially or completely destroyed or recreated revealed that the two stem-loop structures SL1 and SL2 at the 3' end and the poly(A) stretch are necessary for this binding. The interacting nucleotides in such an interaction were further identified by generating footprints of the complex by Pb(II)-induced hydrolysis. This specific binding of viral RdRp to the 3' end of HEV RNA directs the synthesis of complementary-strand RNA and thus such a binding domain might assume the role of a possible cis-acting element as a potential site for the initiation of replication.     

Evidence for equimolar synthesis of double-strand RNA and minus-strand RNA in rotavirus-infected cells

The genome of the rotaviruses consists of eleven segments of double-strand RNA (dsRNA). Each segment is replicated asymmetrically with viral plus-strand RNA, i.e. messenger (m)RNA, serving as the template for the synthesis of minus-strand RNA to produce dsRNA. To examine the relative frequency of replication of each of the eleven genome segments, MA104 cells were infected with low (3rd) and high (12th) passage stocks of simian rotavirus SA11. The total cytoplasmic RNA of the infected cell was radiolabeled either by maintaining the infected cells in the presence [3H]uridine prior to harvest or by 3'-endlabeling the purified RNA with [32P]pCp and T4 RNA ligase. The RNA was then analyzed for the presence of 3H- and 32P-labeled dsRNA by electrophoresis on 10% polyacrylamide gels. Total cytoplasmic RNA from infected cells was also 3'-end-labeled with [32P]pCp and T4 RNA ligase and examined for the presence of minus-strand RNA by electrophoresis on low pH agarose-urea gels. Bands representing dsRNAs and minus-strand RNAs on autoradiographs of the gels were analyzed for intensity by densitometry. The results showed that the eleven segments of viral dsRNA were present in equimolar concentrations in cells either infected with low or high passage stocks of virus. Like intracellular dsRNAs, full-length minus-strand RNAs were also present in equimolar concentration in cells either infected with low or high passage rotavirus. These data indicate that, despite the non-equimolar levels of viral RNAs in the cell, the eleven genome segments of rotavirus are replicated with equal frequencies in vivo.     

A genome-activating N-terminal coat protein peptide of alfalfa mosaic virus is able to activate infection by RNAs 1, 2 and 3 but not by RNAs 1 and 2. Further support for the messenger release hypothesis

Summary. An N-terminal genome-activating peptide of 25 amino acid residues of alfalfa mosaic virus coat protein was unable to activate the incomplete viral genome consisting of RNAs 1 and 2. The messenger release hypothesis predicts that RNA 3 must complement such an inoculum in order to produce RNA 4 that will trigger the process. This is shown indeed to be the case. Received June 6, 2001 Accepted August 6, 2001     

Replication-independent expression of genome components and capsid protein of brome mosaic virus in planta: a functional role for viral replicase in RNA packaging

To begin elucidation of the relationship between Brome mosaic virus (BMV) replication and encapsidation, we used a T-DNA-based Agrobacterium-mediated transient expression (agroinfiltration) system in Nicotiana benthamiana leaves to express either individual or desired pairs of the three genomic RNAs. The packaging competence of these RNAs into virions formed by the transiently expressed coat protein (CP) was analyzed. We found that in the absence of a functional replicase, assembled virions contained non-replicating viral RNAs (RNA1 or RNA2 or RNA3 or RNA1 + RNA3 or RNA2 + RNA3) as well as cellular RNAs. By contrast, virions assembled in the presence of a functional replicase contained only viral RNAs. To further elucidate the specificity exhibited by the functional viral replicase in RNA packaging, replication-defective RNA1 and RNA2 were constructed by deleting the 3' tRNA-like structure (3' TLS). Co-expression of TLS-less RNA1 and RNA2 with wt RNA3 resulted in efficient synthesis of subgenomic RNA4. Virions recovered from leaves co-expressing TLS-less RNA1 and RNA2 and either CP mRNA or wt RNA3 exclusively contained viral RNAs. These results demonstrated that packaging of BMV genomic RNAs is not replication dependent whereas expression of a functional viral replicase plays an active role in increasing specificity of RNA packaging.