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     

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.     

A single amino acid substitution at N-terminal region of coat protein of turnip mosaic virus alters antigenicity and aphid transmissibility

Summary The antigenic activity of the N-terminal region of coat protein of turnip mosaic virus (TuMV) aphid transmissible strain 1 and non-transmissible strain 31 was examined by using a panel of monoclonal antibodies (MAbs) raised against the two virus strains as well as antisera raised against several synthetic peptides from the N-terminal region of the protein. The reactivity of these antibodies was tested in ELISA and in a biosensor system (BIAcore Pharmacia) using virus particles, dissociated coat protein and synthetic peptides as antigens. Substitution of a single amino acid at position 8 in the coat protein of TuMV strain 1 abolished any cross-reactivity between MAbs to strain 1 and the substituted peptide (strain 31) in ELISA although some cross-reactivity was apparent in BIAcore inhibition experiments. In reciprocal tests with MAbs to strain 31 no cross-reactivity with the heterologous peptide was detected in either type of assay. The amino acid residue present at position 8 appears to play a critical role in the binding capacity of MAbs specific for the N-terminal region of TuMV. Antiserum to a synthetic peptide corresponding to residues 1–14 of the protein of TuMV strain 1 was found to react strongly with dissociated coat protein and intact virus particles and was able to inhibit the aphid transmission of the virus. Antiserum to the corresponding peptide of strain 31 did not have this capacity.     

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.     

The cell-free synthesis and assembly of viral specific polypeptides into TMV particles

In a cell-free system derived from wheat germ, the ribonucleic acid of the tobacco mosaic virus mutant Ni 568 directs the synthesis of polypeptides with molecular weights from 10,000 to 140,000, but there is little synthesis of a product equal in size to the coat protein. However, further incubation of the cell-free products with or without extracts from tobacco leaves resulted in the appearance of ³⁵S methionine labeled polypeptides comigrating with tobacco mosaic virus coat protein on sodium dodecyl sulfate-acrylamide gels. Tobacco mosaic virus particles assembled in the presence of these treated cell-free products were shown to be radioactive and co-banded with authentic tobacco mosaic virus during equilibrium centrifugation in cesium chloride. Upon dissociation the assembled particles were found to contain several labeled polypeptides, the most prominent of which comigrated with tobacco mosaic virus coat protein on sodium dodecyl sulfate-acrylamide gels. Furthermore tryptic digests of these polypeptides contained a ³⁵S-methionine labeled peptide with a mobility during high voltage paper ionophoresis at pH 6.5 precisely coincident with that of the only methionine-containing peptide in the coat protein.     

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.     

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.     

Molecular characterization and interviral relationships of a flexuous filamentous virus causing mosaic disease of sugarcane (Saccharum officinarum L.) in India

Summary.  A virus isolate causing mosaic disease of commercial sugarcane was purified to homogeneity. Electron microscopy revealed flexuous filamentous virus particles of ca 890 × 15 nm. The virus isolate reacted positively with heterologous antiserum to narcissus latent virus form UK, but failed to react with potyvirus group specific antiserum. N-terminal sequencing of the intact coat protein (CP) and the tryptic peptides indicated that the virus was probably a potyvirus but distinct from several reported potyviruses. Comparison of the 3′-terminal 1084 nucleotide sequence of the RNA genome of this virus revealed 93.6% sequence identity in the coat protein coding region with the recently described sugarcane streak mosaic virus (Pakistani isolate). The molecular weight of the coat protein (40 kDa) was higher than that deduced from the amino acid sequence (34 kDa). The apparent increase in size was shown to be due to glycosylation of the coat protein which has not been reported thus far in the family, Potyviridae. This is the first report on the molecular characterization of a virus causing mosaic disease of sugarcane in India and the results demonstrate that the virus is a strain of sugarcane streak mosaic virus, a member of the Tritimovirus genus of the Potyviridae. We have named it sugarcane streak mosaic virus – Andhra Pradesh isolate (SCSMV-AP). Received October 14, 1997 Accepted August 7, 1998     

The coat protein genes of squash mosaic virus: cloning,sequence analysis,and expression in tobacco protoplasts

Summary Complementary DNA of the middle-component RNA of the melon strain of squash mosaic comovirus (SqMV) was cloned. Clones containing the coat protein genes were identified by hybridization with a degenerate oligonucleotide synthesized according to the amino acid sequence of a purified peptide fragment of the SqMV large coat protein. A clone containing of 2.5 kbp cDNA insert of SqMV M-RNA was sequenced. The total insert sequence of 2510 bp included a 2373 bp open reading frame (ORF) (encoding 791 amino acids), a 123 bp 3-untranslated region, and a poly(A) region. This ORF is capable of encoding both the 42 and 22 k SqMV coat proteins. Direct N-terminal sequence analysis of the 22 k coat protein revealed its presence at the 3 end of this ORF and the position of the proteolytic cleavage site (Q/S) used to separate the large and small coat proteins from each other. A putative location of the N-terminal proteolytic cleavage site of the 42 k coat protein (Q/N) was predicted by comparisons with the corresponding coat proteins of cowpea mosaic virus, red clover mottle virus, and bean-pod mottle virus. Although the available nucleotide sequences of these viruses revealed little similarity, their encoded coat proteins shared about 47% identity. The identity of the encoded 42 k and 22 k peptides was confirmed by engineering the respective gene regions for expression followed by transfer into tobacco protoplasts using the polyethylene glycol method. Both SqMV coat proteins were expressed in vivo as determined by their reactivity to SqMV coat protein specific antibodies.     

N-terminal basic amino acids of alfalfa mosaic virus coat protein involved in the initiation of infection

Alfalfa mosaic virus coat protein or its messenger RNA is required in the inoculum for virus infection. The N-terminus of the coat protein is required for activity; thus, changes were made in the amino acid sequence of this region. Six coat protein mutants were tested for activity in virus infection assays in protoplasts. A coat protein mutant in which N-terminal residues 3-19 were absent was inactive; whereas, a mutant in which residues 3-11 were absent (CP deltaN9) still had 73% of wild-type activity. Substitution of alanine for the basic residues at positions 14, 17, and 18 in full-length coat protein and in CP deltaN9 resulted in mutant proteins that were inactive in infection. Thus, one, two, or three of these basic residues in CP are required for activity.     

Nucleotide sequence of the 3'-terminal region of potato virus A RNA.

The sequence of the 3'-terminal region of the genome of the potato virus A (PVA) was obtained from two independent cDNA clones. This sequence is 1383 nucleotides long and contains an open reading frame of 1178 nucleotides, ending with the translation termination codon TAA and followed by untranslated region of 205 nucleotides. Since the N-terminal amino acid of the coat protein of PVA was blocked, the position of the putative coat protein cleavage site has been deduced by searching for consensus sequences and by the analogy to other potyviruses. The resulting coat protein is 269 amino acids long and has a calculated MW of 30257. Two independently sequenced cDNA clones show sequence heterogeneity at four nucleotide positions: C422/A422, G432/A432, G446/A446 and T706/C706. Three first nucleotide differences are located at the PVA coat protein N-terminal region and led to the change of the amino acid. The coat protein of PVA displayed significant (73-78%) sequence homology to the coat proteins of six other potyviruses: papaya ringspot virus (PRV), pepper mottle virus (PeMV), plum pox virus (PPV), potato virus Y (PVY), sugarcane mosaic virus (SCMV) and tobacco vein mottling virus (TVMV). Even higher sequence homology (82%) was detected with a coat protein of a seventh potyvirus, tobacco etch virus (TEV). Major differences among the coat protein of PVA and of other potyviruses are located at the N-terminal region of the coat protein.     

Cowpea aphid borne mosaic virus-Morocco and South AfricanPassiflora virus are strains of the same potyvirus

Summary High performance liquid chromatography (HPLC) profiles of tryptic peptides and partial amino acid sequence analysis have been employed to establish the taxonomic status of the Moroccan isolate of cowpea aphid-borne mosaic virus (CABMV). Some previous reports have suggested CABMV to be very closely related to blackeye cowpea mosaic virus (B1CMV) while other reports have concluded that this relationship is distant. In this report a tryptic digest of the coat protein of CABMV-Morocco was compared with those of the coat proteins of B1CMV-Type, B1CMV-W, the mild mottle strain of peanut stripe virus (PStV-MM) and the NY15 strain of bean common mosaic virus (BCMV-NY15), all of which are now recognised as strains of BCMV. The comparisons also included the NL-3 strain of bean necrosis mosaic virus (BNMV-NL3), which had previously been classified as a strain of BCMV. The HPLC peptide profiles indicated that CABMV-Morocco was distinct from BCMV and BNMV. Amino acid sequence analysis of peptides accounting for more than half of the coat protein confirmed that CABMV-Morocco was not a strain of BNMV or BCMV but was a distinct member of the BCMV subset of viruses that previously has been shown to include BCMV, BNMV, soybean mosaic virus, zucchini yellow mosaic virus, passionfruit woodiness virus and South AfricanPassiflora virus (SAPV). Comparison of the partial sequence data with these and other published sequences revealed that the coat protein of CABMV-Morocco is very similar to that of SAPV suggesting that they are strains of the same virus. Since CABMV was described over 25 years earlier than SAPV, the name CABMV should take precedence and SAPV should be renamed CABMV-SAP, the South AfricanPassiflora strain of CABMV.     

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.     

Coat protein of potyviruses 7. Amino acid sequence of peanut stripe virus

Summary The amino acid sequence of the 287-residue coat protein of peanut stripe virus (PStV) was determined from the sequences of overlapping peptide fragments. Results indicated that the amino terminus was blocked by an acetyl group, as has previously been found for the coat protein of Johnsongrass mosaic potyvirus. Comparison of the PStV sequence with coat proteins of 20 distinct potyviruses gave sequence identities of 47–57%, except for zucchini yellow mosaic virus (ZYMV), passionfruit woodiness virus (PWV), and the related strains watermelon mosaic virus 2 (WMV 2) and soybean mosaic virus-N, which showed sequence identities of 70–76%. Several amino acid residues which were common to the core sequences of these coat proteins were at positions previously found to be invariant among potyvirus coat proteins. The degree of these similarities suggests that although PStV, WMV 2, ZYMV, and PWV are distinct potyviruses, they share a common ancestor in their evolutionary development.     

Coat protein of Potyviruses

Summary The amino acid sequence of the coat protein of watermelon mosaic virus 2 (WMV 2) was determined by a combination of peptide and nucleic acid sequencing. The coat protein of WMV 2 contained 281 amino acid residues including a single cysteine at position 132 and a blocked amino terminus. Comparison with the coat protein sequences of 20 strains of ten distinct potyviruses showed sequence homologies ranging from 43% to 69% except for the N strain of soybean mosaic virus (SMV-N), where the sequence homology with WMV 2 was 83%. This degree of homology and the location of sequence differences between WMV 2 and SMV-N is much closer to that observed between strains of the same virus than that found between distinct potyviruses. These data suggest that WMV 2 and SMV-N may be strains of the same virus.     

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.     

Watermelon mosaic virus-Morocco is a distinct potyvirus

Summary The relationship of the Morocco isolate of watermelon mosaic virus (WMV) to WMV 2, soybean mosaic virus (a virus closely related to WMV 2) and the W strain of papaya ringspot virus (PRSV-W), formerly WMV 1, was examined by comparing tryptic peptide profiles using high performance liquid chromatography. The profiles indicated that the coat protein sequence of WMV-Morocco differed substantially from those of the other potyviruses. This conclusion was supported by sequence data from five tryptic peptides from the coat protein of WMV-Morocco, which showed only 61–68% identity to equivalent sequences in PRSV-W, WMV 2 and zucchini yellow mosaic, another potyvirus infecting cucurbits. Based on the above data, and on known correlations between coat protein sequence similarities and potyvirus relationship, it is concluded that WMV-Morocco should be regarded as a distinct potyvirus.     

Limited sequence variation in the leader sequence of RNA 4 from several strains of alfalfa mosaic virus

The sequences of the 5'-noncoding regions of RNA 4 of seven strains of alfalfa mosaic virus were compared. In each case, the coat protein cistron was preceded by a leader sequence of 39 nucleotides (including the initiator codon). At position 26, an A-, G-, or U-residue was found. Otherwise the leader sequences are identical.