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.     

Evidence that alfalfa mosaic virus infection starts with three RNA-protein complexes

The tripartite genome of alfalfa mosaic virus (AMV) needs to be activated by its coat protein. To establish whether coat protein exerts its role by interacting structurally with one, two, or all three AMV-RNA species, the infectivity of mixtures of RNA-protein complexes with free RNAs were studied (Smit and Jaspars, Virology 104, 454-461, 1980). These studies were not fully conclusive since some redistribution of coat protein does occur as soon as RNAs are brought into contact with RNA-protein complexes. This problem was overcome by the use of ts mutants of AMV. Free ts coat protein subunits were not able to activate the wt genomic RNAs at 30 degrees in tobacco. Once complexed to the genomic RNAs at 0 degrees , the biological activity of the ts coat protein remained when assayed at the nonpermissive temperature. Apparently, the ts coat protein is inactivated during attempted redistribution at 30 degrees . Studies of mixtures of RNA-protein complexes and free RNA show that coat protein has to be present on at least two of the three RNA species. This result in combination with previous results (Smit and Jaspars, 1980) warrants the conclusion that the alfalfa mosaic virus infection starts with three RNA-protein complexes.     

Limited proteolysis of alfalfa mosaic virus: influence on the structural and biological function of the coat protein

Limited tryptic digestion of intact alfalfa mosaic virus resulted in the quantitative removal of 27 amino acids from the N-terminal portions of the coat protein subunits. The release of this peptide material, which contains a relatively high number of basic residues, causes a breakdown of the bacilliform viral components into spherical nucleoprotein particles. From this it was concluded that the proteolytic cleavage interferes with protein-RNA interactions in the virus, but not with protein-protein interactions. The release of the N-terminal peptide also destroyed the capacity of the coat protein to activate the alfalfa mosaic virus genome. This supports the hypothesis that this activation is accomplished by a specific interaction of the coat protein with alfalfa mosaic virus RNAs.     

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.     

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.     

Further implications for the evolutionary relationships between tripartite plant viruses based on cucumber mosaic virus RNA 3

The nucleotide sequence of the RNA 3 of the Q-strain of cucumber mosaic virus (Q-CMV) has been reinvestigated and supporting partial amino acid sequence data obtained for the coat protein. Corrections to the previously published sequence of RNA 3 [A. R. Gould and R. H. Symons (1982) Eur. J. Biochem. 126, 217-226] result in changes to the size and composition of the putative 3a and coat proteins. Analysis of the nucleotide sequence revealed a 14-nucleotide sequence present in the intercistronic regions of the RNA 3 molecules of both Q-CMV and brome mosaic virus (BMV). This sequence, which is closely related to sequences previously detected in the 5'-untranslated region of Q-CMV and BMV RNAs 1 and 2 [M. A. Rezaian, R. H. V. Williams, and R. H. Symons (1985) Eur. J. Biochem. 150, 331-339], may be important in the control of RNA synthesis. Computer-assisted comparisons indicate an ancestral relationship between the 3a proteins of CMV, BMV, and alfalfa mosaic virus (AMV) and between the coat proteins of CMV and BMV. These comparisons significantly extend previous observations regarding the close evolutionary relationships within the plant tripartite virus group.     

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.     

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%.     

Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo

Brome mosaic virus (BMV) is an icosahedral virus with a tripartite RNA genome which infects monocotyledonous plants, while the cowpea or legume strain of tobacco mosaic virus (CcTMV) is a rod-shaped virus with a single component RNA genome which infects dicotyledonous plants. To examine the potential for exchanging entire genes between RNA viruses, biologically active cDNA clones were used to replace the natural coat gene of BMV RNA3 with the coat gene and encapsidation origin of CcTMV. In protoplasts coinoculated with BMV RNAs 1 and 2, the resulting hybrid RNA3 was replicated by BMV trans-acting factors but was packaged in TMV coat protein to give rod-shaped particles rather than the usual BMV icosahedra. When the CcTMV encapsidation origin was suitably inserted in derivatives of BMV RNAs 1 and 2, these RNAs were also packaged in a ribonuclease-resistant form in protoplasts coinoculated with the hybrid RNA3 expressing TMV rather than BMV coat protein. Thus, despite the markedly divergent nature of BMV and TMV, replicating hybrids bearing characters derived from both parent viruses were produced. Such hybrid viruses could be of considerable value for studying specific steps in infection and for assigning functions to particular virus genes.     

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     

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.     

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.     

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     

Mapping of the two coat protein genes on the middle RNA component of squash mosaic virus (comovirus group)

Squash mosaic virus, a member of the comovirus group, has a divided genome identified as middle-component RNA (M RNA; MW = 1.4 x 10(6)) and bottom-component RNA (B RNA; MW = 2 x 108). The isometric capsid of squash mosaic virus is composed of two distinct protein monomers with molecular weights of 22,000 (22k) and 42k. The isolated RNA components were translated in a rabbit reticulocyte lysate system. Translation products of the B RNA had estimated molecular weights of 190k, 51k, and 32k, while the M RNA products ranged in estimated molecular weight from 22k to 112k. Products of the B RNA did not react with antisera prepared to the 22k and 42k coat proteins. All of the M RNA products reacted with antiserum to the 22k coat protein and all the products larger than 35k reacted with antiserum to the 42k coat protein. The 22k product of M RNA translation had a V-8 protease peptide pattern identical to that of the 22k coat protein. Protease peptide patterns of the M RNA 64k and 112k-105k translation products showed a number of peptide fragments similar to those produced by the 22k and 42k coat proteins, indicating that the translation products contained the sequences of the two coat proteins. The proposed gene order of translation for squash mosaic virus M RNA is as follows: 5' end-22k coat protein gene-42k coat protein gene-48k unidentified protein gene-untranslated sequence-3' end.     

A Core Promoter Hairpin is Essential for Subgenomic RNA Synthesis in Alfalfa Mosaic Alfamovirus and is Conserved in other Bromoviridae

The nucleotide sequence immediately in front of the initiation site for subgenomic RNA 4 synthesis on RNA 3 minus strand, which has been proved to function as a core promoter, was inspected for secondary structure in 26 species of the plant virus family Bromoviridae. In 23 cases a stable hairpin could be predicted at a distance of 3 to 8 nucleotides from the initiation site of RNA 4. This hairpin contained several conserved nucleotides that are essential for core promoter activity in brome mosaic virus (R.W. Siegel, S. Adkins and C.C. Kao, Proc. Natl. Acad. Sci. USA 94, 11238–11243, 1997). Phylogenetic evidence and evidence from the effect of artificial mutations reported in the literature (E.A.G. van der Vossen, T. Notenboom and J.F. Bol, Virology 212, 663–672, 1995) indicate that the stem-loop structure is essential for promoter activity in alfalfa mosaic virus and probably in other Bromoviridae. Stability of the hairpin is most pronounced in the genera Alfamovirus and Ilarvirus which display genome activation by coat protein. The hypothesis is put forward that with these viruses the coat protein is needed for the viral RNA polymerase to interact with the core promoter hairpin leading to access for the enzyme to the initiation site of RNA 4.     

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     

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.