Brome mosaic virus coat protein inhibits viral RNA synthesis in vitro

In vitro, the coat protein of brome mosaic virus (BMV) inhibited RNA synthesis by replicase extracted from BMV-infected barley leaves. The inhibition was due to the interaction of coat protein with BMV RNA. Under the same conditions, no inhibition by TMV coat protein or bovine serum albumin was detected. For the complete inhibition of RNA synthesis in vitro, a coat protein:RNA ratio of 620:1 was required, and their preincubation before addition to the reaction mixture was essential. If the coat protein was added to the reaction mixture during incubation, synthesis continued for a few minutes at the level of the control (omission of coat protein), then decreased gradually, and stopped 6 min after the addition of coat protein. These results suggest that the inhibitory effect of coat protein on RNA synthesis in vitro is due to the interference with the binding site of replicase by partial reassembly of nucleoprotein and that this phenomenon may be a cause of cross protection.     

Regulation of single-strand RNA synthesis of alfalfa mosaic virus in non-transgenic cowpea protoplasts by the viral coat protein

Summary.  We have compared the RNA synthesis of alfalfa mosaic virus in complete (by RNAs 1, 2 and 3) and incomplete infections (by RNAs 1 and 2) of cowpea protoplasts. Both viral RNA polymerase activity and accumulation of viral RNA were measured. By annealing RNA in solution with ³²P-labelled probes of plus and minus polarity followed by treatment with ribonucleases, we determined viral RNAs quantitatively in both single- and double-stranded RNA fractions. The accumulation of single-stranded RNA of positive polarity differed considerably between the two types of infection (250 ng vs. less than 1 ng per 10⁵ protoplasts), although viral RNA polymerase activities as measured in vitro and the concentrations of minus RNA were similar. Since the method also measured fragmented RNA, this difference is probably not due to lack of protection of viral RNA by coat protein during incomplete infection. Synthesis of single-stranded plus RNA requires either RNA 3 itself or one of its gene products. We postulate that coat protein is the stringent regulator of alfalfa mosaic virus genomic expression. Accepted November 3, 1997 Received August 14, 1997     

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.     

Specificity of RNA and coat protein interaction in alfalfa mosaic virus and related viruses

Well-defined coat protein binding sites were found to be present on the genomic RNAs of AlMV and TSV. In view of the regulatory importance of these sites in virus multiplication, the possibility that nonrelated proteins also were able to interact with these sites was investigated. The coat proteins of TSV, BMV, CMV, and SBMV recognize specific sites on AlMV RNA 1. The significance of these sites in virus multiplication is discussed. No specific binding sites were found with TMV coat protein or the nonviral proteins ovalbumin, myoglobin, and lysozyme. Moreover, the ability of AlMV coat protein to recognize specific sites on the heterologous RNAs of TSV, BMV, bacteriophage MS2, and Escherichia coli was tested. No specific sites were found with MS2-RNA. However, specific binding sites were found with BMV-RNA 3 and, unexpectedly, with E. coli 16 S ribosomal RNA. From these data it was concluded that binding of AlMV and TSV coat proteins to their genomic RNAs is a specific process. However, the binding of coat protein to BMV-RNAs and to ribosomal RNAs may result from secondary folding that may have been conserved for other purposes throughout evolution of the RNAs.     

Minimum requirements for specific binding of RNA and coat protein of alfalfa mosaic virus

Coat protein-protected fragments of alfalfa mosaic virus RNA (AlMV-RNA) and tobacco streak virus RNA (TSV-RNA), which were isolated as described [D. Zuidema, M. F. A. Bierhuizen, B. J. C. Cornelissen, J. F. Bol, and E. M. J. Jaspars (1983)Virology, 125, 361-369], were tested for their ability to rebind AlMV coat protein in the presence of an excess of Escherichia coli tRNA by means of a nitrocellulose filter retention assay. In order to obtain the minimum requirements for coat protein binding, a 3'-terminal binding site and several internal binding sites were isolated and fragmented by mild alkali treatment so that various lengths of a particular binding site were present in the mixture to be tested for rebinding capacity. All fragments which originated from the Wend of AlMV-RNA 1 and could bind AlMV coat protein have in common the sequence 5'-CUCAUGCUA-3'. However, this sequence alone is not sufficient to bind viral coat protein. Either an extension by at least 27 nucleotides of this oligomer to the right or an extension by 45 nucleotides (or possibly less) to the left is necessary for AlMV coat protein binding. Also, smaller extensions simultaneously occurring at both sides are sufficient. The smallest fragment which still has binding capacity for viral coat protein is 23 nucleotides long and originates from an internal site of RNA 1. All bound fragments have two common features: the occurrence of AUG(C) twice in the sequence and the potential ability to form a stable secondary structure. A striking observation was that 3'-terminal fragments of TSV-RNAs 1 and 2 rebind AlMV coat protein with low efficiency (about 27 and 37%, respectively), whereas a 3'-terminal fragment of TSV-RNA 3 rebinds AlMV coat protein with an efficiency of about 71%.     

Subcellular localization of tobacco mosaic virus minus strand RNA in infected protoplasts

Radioactive RNA probes were prepared which specifically hybridize with sequences complementary to 5' and 3' regions of tobacco mosaic virus (TMV) RNA. These probes were used in Northern hybridization to locate TMV-RNA minus strands in the subcellular fractions of infected tobacco protoplasts. When the protoplasts were lysed with Triton X-100, full-length minus strands were present in the cytoplasmic but not in the nuclear fraction. With mechanically broken protoplasts, the crude nuclear fraction (250 g pellet) contained small amount of minus strands which appeared to derive from unbroken protoplasts, but most of minus strands were recovered in a fraction sedimented between 250 and 2500 g, little if any being found in lighter fractions. The results indicate that TMV-RNA replicates in association with an extranuclear structure.     

Genetic dissection of the multiple functions of alfalfa mosaic virus coat protein in viral RNA replication, encapsidation, and movement

Coat protein (CP) of alfalfa mosaic virus (AMV) binds as a dimer to the 3' termini of the three genomic RNAs and is required for initiation of infection, asymmetric plus-strand RNA accumulation, virion formation, and spread of the virus in plants. A mutational analysis of the multiple functions of AMV CP was made. Mutations that interfered with CP dimer formation in the two-hybrid system had little effect on the initiation of infection or plus-strand RNA accumulation but interfered with virion formation and reduced or abolished cell-to-cell movement of the virus in plants. Six of the 7 basic amino acids in the N-terminal arm of CP (positions 5, 6, 10, 13, 16, and 25) could be deleted or mutated into alanine without affecting any step of the replication cycle except systemic movement in plants. Mutation of Arg-17 interfered with initiation of infection (as previously shown by others) and cell-to-cell movement of the virus but not with plus-strand RNA accumulation or virion formation. The results indicate that in addition to the RNA-binding domain, different domains of AMV CP are involved in initiation of infection, plus-strand RNA accumulation, virion formation, cell-to-cell movement, and systemic spread of the virus.     

Virus protein synthesis in alfalfa mosaic virus infected alfalfa protoplasts

Four proteins unique to virus infection were synthesized in alfalfa mosaic virus-infected alfalfa mesophyll protoplasts. These proteins, P1, P2, P3, and coat protein comigrated on electrophoresis with the major in vitro translation products of RNA 1, RNA 2, RNA 3, and RNA 4, respectively. P1, P3, and coat protein were observed at 5 hr post inoculation; P2 was detected at 9 hr post inoculation. The three nonstructural proteins accumulated most rapidly early in infection until about 15 hr post inoculation; stable protein levels were maintained thereafter. Coat protein accumulated rapidly until about 20 hr after inoculation. All four virus RNA species were detected in infected protoplasts by labelling with [3H]uridine. Ultraviolet irradiation of protoplasts prior to inoculation was necessary for virus protein detection, but it severely depressed the synthesis of RNA 1 and RNA 2 relative to RNA 3 and RNA 4.     

The location of coat protein and viral RNAs of alfalfa mosaic virus in infected tobacco leaves and protoplasts

The location of coat protein of alfalfa mosaic virus (AIMV) strain 425 was determined in protoplasts isolated from infected tobacco leaves and in in vitro inoculated tobacco protoplasts, using immunocytochemistry on ultrathin frozen sections labeled with colloidal gold. In infected tobacco leaves 5 days postinoculation (p.i.) coat protein is present in the cytoplasm and nucleus, especially around the nucleolus. In in vitro inoculated tobacco protoplasts coat protein was not present in the nucleus 6 hr p.i. These results indicate that the presence of coat protein in the nucleus is not necessary for viral replication. However, coat protein could be detected in the nucleus 48 hr p.i. Probably coat protein or virus particles accumulate in the nucleus late in infection. Minus-strand RNA, as part of the replication complex, could be detected in a 650 g pellet fraction of infected tobacco leaves but not in the nucleus, suggesting that replication of AIMV occurs outside the nucleus.     

Mechanism of synthesis of turnip yellow mosaic virus coat protein subgenomic RNA in vivo

Turnip yellow mosaic virus (TYMV) possesses a monopartite single-stranded (+) sense RNA genome in which the coat protein (cp) gene is 3' proximal and is expressed in vivo via a subgenomic RNA. Evidence is presented here that this subgenomic RNA is synthesized in vivo by internal initiation of replication on (-) RNA strands of genomic length. The double-stranded RNAs (dsRNAs) from TYMV-infected plants have been isolated, purified, and characterized. Under native conditions, no dsRNAs (replicative intermediates and/or replicative forms) of subgenomic length corresponding to subgenomic cp RNA can be detected by ethidium bromide staining of RNA-sizing gels or by Northern blot hybridization using RNA probes. The presence of nascent subgenomic cp (+) RNA strands on the dsRNA of genomic length has been demonstrated using two different approaches: (1) Northern blot hybridization using (-) RNA probes under denaturing conditions and (2) characterization of the 5' ends of nascent (+) RNA strands upon labeling by vaccinia virus nucleoside-2'-methyltransferase.     

Inhibition of alfalfa mosaic virus RNA and protein synthesis by actinomycin D and cycloheximide

Actinomycin D, added early after inoculation, reduces the production of infectious alfalfa mosaic virus in cowpea protoplasts by 90%. This reduction was associated with an inhibition of viral minus-strand and plus-strand RNA synthesis, suggesting the involvement of host factors in these processes. Coat protein production was less affected by the drug. Addition of cycloheximide throughout the growth cycle resulted in an immediate cessation of coat protein production and an enhanced degradation of viral RNA. This degradation obscured possible effects of the drug on viral RNA synthesis.     

Primary structure of alfalfa mosaic virus coat protein (strain S)

This communication describes the sequence of the 217 amino acid residues of the alfalfa mosaic virus (strain S) protein subunit. This protein has a molecular weight of 23,655.     

The importance of alfalfa mosaic virus coat protein dimers in the initiation of replication

Deletion and substitution mutations affecting the oligomerization of alfalfa mosaic virus (AMV) coat protein (CP) were studied in protoplasts to determine their effect on genome activation, an early step in AMV replication. The CP mutants that formed dimers, CPDeltaC9 and CPC-A(R)F, were highly active in initiating replication with 63-84% of wild-type (wt) CP activity. However, all mutants that did not form dimers, CPDeltaC18, CPDeltaC19, CPC-WFP, and CPC-W, were much less active with 19-33% of wt CP activity. The accumulation and solubility of mutant CPs expressed from a virus-based vector in Nicotiana benthamiana were similar to that of wt CP. Analysis of CP-RNA interactions indicated that CP dimers and CP monomers interacted very differently with AMV RNA 3' ends. These results suggest that CP dimers are more efficient for replication than CP monomers because of differences in RNA binding rather than differences in expression and accumulation of the mutant CPs in infected cells.     

Messenger RNA for the coat protein of southern bean mosaic virus

When RNA from southern bean mosaic virus is fractionated on a sucrose gradient, the resulting absorbance profile shows a major peak corresponding to 1.4 x 10(6) MW together with a considerable amount of slower sedimenting material. The RNA from these gradient fractions was translated in wheat embryo extracts and reticulocyte lysates. Only RNA in the molecular weight range 0.3 - 0.4 x 10(6) was found to induce synthesis of coat protein (designated P3). RNA of molecular weight 1.4 x 10(6) induced synthesis of three proteins, P1, P2, and P4.     

Alfalfa mosaic virus coat protein bridges RNA and RNA-dependent RNA polymerase in vitro

Alfalfa mosaic virus (AMV) RNA replication requires the viral coat protein (CP). AMV CP is an integral component of the viral replicase; moreover, it binds to the viral RNA 3'-termini and induces the formation of multiple new base pairs that organize the RNA conformation. The results described here suggest that AMV coat protein binding defines template selection by organizing the 3'-terminal RNA conformation and by positioning the RNA-dependent RNA polymerase (RdRp) at the initiation site for minus strand synthesis. RNA-protein interactions were analyzed by using a modified Northwestern blotting protocol that included both viral coat protein and labeled RNA in the probe solution ("far-Northwestern blotting"). We observed that labeled RNA alone bound the replicase proteins poorly; however, complex formation was enhanced significantly in the presence of AMV CP. The RNA-replicase bridging function of the AMV CP may represent a mechanism for accurate de novo initiation in the absence of canonical 3' transfer RNA signals.