Removal of the N-terminal part of alfalfa mosaic virus coat protein interferes with the specific binding to RNA 1 and genome activation

Trypsinized coat protein of alfalfa mosaic virus lacking 25 amino acids at its N terminus still has the capability to form complexes with RNA which are detectable by sedimentation in sucrose gradients. However, it does not protect specific sites on the RNA against degradation by ribonuclease, as the native coat protein does (D. Zuidema, M. F. A. Bierhuizen, B. J. C. Cornelissen, J. F. Bol, and E. M. J. Jaspars (1983) Virology 125, 361-369.). The trypsinized coat protein has lost the capacity of the native coat protein to make the genome RNAs of alfalfa mosaic virus infectious or to interfere with the infectivity brought about by the native coat protein. These findings suggest that genome activation occurs via binding of the N-terminal part of the coat protein to specific sites on the RNAs.     

The effect of inhibitors of protein biosynthesis on the infectivity of alfalfa mosaic virus: role of coat protein

Cycloheximide, when present in the inoculum at a concentration of 50 μg/ml decreases the infectivity of nucleoproteins of strain 425 of alfalfa mosaic virus (AMV) by more than 90%. Infectivity of the nucleoproteins of the AMV strain yellow spot mosaic virus (YSMV) and the Strasbourg strain were much less sensitive to cycloheximide; at 50 μg/ml of the antibiotic 60–80% of the normal infectivity was found. However, when chloramphenicol and cycloheximide were given simultaneously, the infectivity of these strains was as much reduced as that of AMV 425 in the presence of cycloheximide alone. As was shown earlier, infectious RNA preparations consist of 4 RNA species, 3 large RNAs constituting the complete genome, and a small monocistronic RNA, the top component a RNA. When the latter is removed, the RNA preparation is no longer infectious. A mixture of bottom, middle, and top component b RNAs can be activated by the coat protein. The infectivity of the 4 RNAs from YSMV and AMV 425 was equally sensitive to cycloheximide. A combination of YSMV RNA activated by AMV 425 coat protein was as sensitive to cycloheximide as AMV 425 nucleoprotein. This suggests that the coat protein plays a role in the localization of translation, which is in accordance with the previous finding that sensitivity to cycloheximide is determined by the top component b RNA, which contains the genetic information for the coat protein.     

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.     

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.     

The host DNA sequences in different populations of serially passaged SV40

The RNAs constituting the genome of alfalfa mosaic virus (AMV) have been separated by electrophoresis on polyacrylamide gels and inoculated alone or in mixture upon susceptible plants. The isolation and analysis of replicative forms (RF) found in these plants revealed that the mixtures which induce the formation of RF are the same as those which give rise to local lesions on hypersensitive plants: that is, only the mixture of 4 species of AMV RNA, or the mixture of the 3 largest RNAs in addition to coat protein is able to induce the formation of RF, whereas no single RNA alone or with the addition of small amounts of coat protein gives rise to the corresponding replicative form.The problem of the existence of a replicative form of 12 S RNA was also examined. It was never possible to show any replicative form of 12 S RNA even when a strain producing much 12 S RNA (AMV₄₂₅) was used. Some possibilities of 12 S RNA biosynthesis were examined.     

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.     

Amino acid analysis of alfalfa mosaic virus coat proteins: An aid for viral strain identification

Amino acid analysis of the coat proteins from different strains of alfalfa mosaic virus (AMV) can be used as a tool for strain characterization. This method together with carboxy-terminal amino acid analysis of 11 different AMV strains allowed three groups of closely related AMV coat proteins to be distinguished. A rough estimation of the evolutionary relationships among these coat proteins is presented. New chemical evidence is provided in support of previous genetic experiments that have localized the coat protein cistron on the Tb-component RNA.     

Specificity of in vitro reconstitution of bromegrass mosaic virus

Bromegrass mosaic virus (BMV) RNA was allowed to compete with yeast tRNA or alfalfa mosaic virus (AMV) RNA for in vitro encapsidation by BMV protein. Various proportions of 32P-labeled foreign RNAs were added to a reassembly mixture (BMV protein: BMV RNA, 4:1) and the reassembly products were observed by analytical and rate-zonal sedimentation, and the RNA contents of the nucleocapsids were examined by gel electrophoresis. Incubation of BMV protein with tRNA alone produced 56 S particles containing five or six tRNA molecules per particle, but with both tRNA and BMV RNA present very little of the tRNA was incorporated. AMV 12 S RNA led to 64 S particles containing one AMV RNA molecule: with both AMV and BMV RNAs present, the smallest BMV RNA outcompeted the AMV RNA about fourfold, even though the two RNAs have similar molecular weights and biological functions. Evidently BMV protein does to some extent specifically recognise its own RNA molecules.     

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.     

Coat protein is required for infectivity of tobacco streak virus: biological equivalence of the coat proteins of tobacco streak and alfalfa mosaic viruses.

The four nucleoprotein components of tobacco streak virus (TSV) were purified by zonal centrifugation. Each component contains mainly one RNA species. Infectivity studies indicate that the three fastest-sedimenting nucleoproteins are required for viral replication. A mixture of the three largest RNA's however, is not infectious but can be activated by the viral coat protein or by the smallest TSV-RNA. The same situation exists in alfalfa mosaic virus (AMV). The coat proteins of TSV and AMV, which are rather different as judged by serology and tryptic digestion products, cannot only activate their own genome but also that of each other. The nucleoprotein components of the two viruses, however, could not be substituted for each other.TSV-RNA is as efficient as AMV-RNA in withdrawing protein subunits from AMV nucleoprotein. However, TSV nucleoprotein could not be uncoated by its own RNA or by AMV-RNA. Nevertheless, the affinity of TSV-RNA for AMV coat protein and the functional equivalence of the two coat proteins point to a possible common origin of these two viruses.     

A mutant of alfalfa mosaic virus with an unusual structure

A spontaneous mutant of alfalfa mosaic virus (AMV) with an altered structure is described. By analysis of pseudorecombinants the mutation(s) responsible for the altered structure were assigned to RNA 3. By in vitro translation and serology it is shown that both proteins (35K protein and coat protein) encoded by RNA 3 are changed. The mutation(s) present in RNA 3 also have an effect on the ratio of the three genomic RNAs, both in virion as well as in double-stranded RNA preparations. Compared to wild-type particles mutant particles (1) have a lower electrophoretic mobility in polyacrylamide gels, (2) have a greater sedimentation velocity in sucrose density gradients, (3) do contain the same percentage of RNA, (4) are more prone to aggregation, (5) are somewhat less stable during storage, and (6) are less sensitive to uncoating by AMV-RNA. Electron micrographs show that the mutant preparations contain some very long bacilliform particles, however the majority of the particles is spheroidal and bear a strong resemblance to the particles of the ilarviruses. The structural properties of this mutant support the classification of AMV as an ilarvirus.     

Evidence that RNA 4 of alfalfa mosaic virus does not replicate autonomously

A mutant (Tbts7) of alfalfa mosaic virus, the coat protein of which is unable to activate the viral genome (the RNA species 1, 2, and 3, which need some coat protein for infectivity) at 30 degrees , can be rescued at this temperature by adding to the inoculum wild-type RNA 3 (the genome part that contains the coat protein cistron), but not adding wild-type RNA 4 (the subgenomic messenger for the coat protein). Unless RNA 3 of Tbts 7 has a second ts mutation at a site not occurring in RNA 4, it may be concluded from the above finding that RNA 4 does not replicate autonomously.     

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.     

Activation of the genome of alfalfa mosaic virus is enhanced by the presence of the coat protein on all three genome parts

The same amount of coat protein stimulates the infectivity of alfalfa mosaic virus RNA more when added to the three genome RNAs at once than when preincubated with one or two genome RNAs separately before the inoculum is completed. This suggests that the coat protein activates the genome by interacting with all three parts of it. It could not be demonstrated that infectivity is absolutely dependent on this multiple activation because of the possible exchange of protein between RNA molecules in the inoculum. However, factors that are likely to influence this exchange also have an effect on infectivity. Experiments showed that complex formation of coat protein with only the smallest genome RNA (that contains the coat protein gene) does not enhance infectivity as compared with other individual RNAs and protein combinations. Apparently the expression of the coat protein gene is not stimulated in this manner.     

Activation of Prunus necrotic ringspot virus and rose mosaic virus by RNa 4 components of some llarviruses

RNA extracted from rose mosaic virus (RMV) and from necrotic ringspot virus-G(NRSV) separated into four sedimenting components in sucrose density gradients. The components were designated RNAs 1, 2, 3, and 4 in order of decreasing sedimentation velocities. Sodium dodecyl sulfate (SDS)-denatured NRSV and RMV preparations showed a prominent protein species of about 25,000 daltons when electrophoresed in polyacrylamide gels. In addition, two closely migrating protein species of about 19,000 daltons were also observed in SDS-denatured RMV preparations. Preparations containing RNA 1+2+3 and preparations containing RNA 4 were obtained by two successive cycles of sucrose density gradient centrifugation. Neither preparation was infectious alone. Mixtures of RNA 1+2+3 from each virus became infectious, however, with the addition of homologous RNA 4. RMV protein also activated RMV-RNA 1+2+3 preparations. NRSV-RNA 4 efficiently activated RMV-RNA 1+2+3, but RMV-RNA 4 had very little ability to activate NRSV-RNA 1+2+3 even though it efficiently activated its homologous RNA 1+2+3 preparations. RNA 4 preparations from alfalfa mosaic virus (AMV) and citrus leaf rugose virus (CLRV) activated RMV-RNA 1+2+3. Comparable concentrations of RMV-RNA 4, however, failed to activate RNA 1+2+3 from AMV and from CLRV. RNA from CLRV, RMV, and citrus variegation virus (CVV) efficiently uncoated AMV particles. However, AMV-RNA and CLRV-RNA 4 uncoated CVV but not CLRV or RMV. The evidence presented indicated that viruses in the new Ilarvirus group [Shepherd, R. J., et al. (1976). Intervirology 6, 181–1841 have similar infectivity requirements; that is, mixtures of RNA 1+2+3 are not infectious but can be activated by either RNA 4 or coat protein.     

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.     

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     

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