Single amino acid substitutions globally suppress the folding defects of temperature-sensitive folding mutants of phage P22 coat protein.

The amino acid sequence of a polypeptide defines both the folding pathway and the final three-dimensional structure of a protein. Eighteen amino acid substitutions have been identified in bacteriophage P22 coat protein that are defective in folding and cause their folding intermediates to be substrates for GroEL and GroES. These temperature-sensitive folding (tsf) substitutions identify amino acids that are critical for directing the folding of coat protein. Additional amino acid residues that are critical to the folding process of P22 coat protein were identified by isolating second site suppressors of the tsf coat proteins. Suppressor substitutions isolated from the phage carrying the tsf coat protein substitutions included global suppressors, which are substitutions capable of alleviating the folding defects of numerous tsf coat protein mutants. In addition, potential global and site-specific suppressors were isolated, as well as a group of same site amino acid substitutions that had a less severe phenotype than the tsf parent. The global suppressors were located at positions 163, 166, and 170 in the coat protein sequence and were 8-190 amino acid residues away from the tsf parent. Although the folding of coat proteins with tsf amino acid substitutions was improved by the global suppressor substitutions, GroEL remained necessary for folding. Therefore, we believe that the global suppressor sites identify a region that is critical to the folding of coat protein.     

A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysine-rich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious.     

Coat protein polymerization of alfalfa mosaic virus strain VRU

The association behaviour of the coat protein of alfalfa mosaic virus strain VRU was studied by sedimentation analysis and electron microscopy. The results of this study were compared with the data obtained from similar studies with the coat protein of strain 425 (Driedonks et al., 1977). In the depolymerized state VRU protein is likely a dinier of the 24,050 molecular weight polypeptide chain. The main association product is a tubular structure with a diameter of about 180 Å. The optimum conditions for the reaction were polyphosphate-containing buffer at pH 6·5. Optical diffraction analysis of negatively stained specimens revealed a helical arrangement of the protein subunits in these assemblies. The same type of reaction product was found when the association reaction was carried out in the presence of polynucleotides. The length of the VRU particles is abnormally long compared to other alfalfa mosaic virus strains. This phenomenon can be ascribed to the tendency of the protein to polymerize into tubular rather than spherical particles.     

Nucleotide sequence analysis of cDNA encoding the coat protein of cucumber mosaic virus: genome organization and molecular features of the protein

The cDNA sequence coding for the coat protein of cucumber mosaic virus (Japanese Y strain) was cloned, and its nucleotide sequence was determined. The sequence contains an open reading frame that encodes the coat protein composed of 218 amino acids. The nucleotide and deduced amino acid sequences of the coat protein of this strain were compared with those of the Q strain; the homologies of the sequences were 78% and 81%, respectively. Further study of the sequences gave an insight into the genome organization and the molecular features of the coat protein. The coding region can be divided into three characteristic regions. The N-terminal region has conserved features in the positively charged structure, the hydropathy pattern and the predicted secondary structure, although the amino acid sequence is varied mainly due to frameshift mutations. It is noteworthy that the positions of arginine residues in this region are highly conserved. Both the nucleotide and amino acid sequences of the central region are well conserved. The amino acid sequence of the C-terminal region is not conserved, because of frameshift mutations, however, the total number of amino acids is conserved. The nucleotide sequence of the 3'-noncoding region is divergent, but it could form a tRNA-like structure similar to those reported for other viruses. Detailed investigation suggests that the Y and Q strains are evolutionarily distant.     

Messenger RNA structure participating in the initiation of synthesis of cucumber mosaic virus coat protein

The sequence of the 5'-terminal 106 nucleotides of cucumber mosaic virus (strain Y) RNA 4, the mRNA coding for viral coat protein, has been determined. The first AUG was located at 77 nucleotides from the 5'-terminus and was confirmed to be an initiation codon by analysis of the N-terminal amino acid sequence of the protein. The nucleotide sequence (positions 77-106) beyond the AUG codon predicted the sequence of ten amino acids corresponding to the N-terminal region of the protein, which exactly matched the determined amino acid sequence containing an acetyl methionine as the N-terminal amino acid. The distance of the initiation codon AUG from the cap structure was 76 nucleotides and the longest among the mRNAs for coat protein of plant viruses so far reported (9-36 nucleotides). This noncoding region is rich in U residues (40%) and the number of G residues (21 nucleotides) is the largest among these mRNAs (usually 1 or 2 residues). A possible secondary structure is postulated for the region, which might be implicated in efficient translation of the RNA 4 in vivo.     

Amino-terminal amino acid sequence of p10, the fifth major gag polypeptide of avian sarcoma and leukemia viruses.

We have identified p10 as a fifth gag protein of avian sarcoma and leukemia viruses. Amino-terminal protein sequencing of this polypeptide purified from the Prague C strain of Rous sarcoma virus and from avian myeloblastosis virus implies that it is encoded within a stretch of 64 amino acid residues between p19 and p27 on the gag precursor polypeptide. For p10 from the Prague C strain of Rous sarcoma virus the first 30 residues were found to be identical with the predicted amino acid sequence from the Prague C strain of Rous sarcoma virus DNA sequence, whereas for p10 from avian myeloblastosis virus the protein sequence for the same region showed two amino acid substitutions. Amino acid composition data indicate that there are no gross composition changes beyond the region sequenced. The amino terminus of p10 is located two amino acid residues past the carboxy terminus of p19, whereas its carboxy terminus probably is located immediately adjacent to the first amino acid residue of p27.     

The amino acid sequence of the cowpea strain of tobacco mosaic virus protein.

The amino acid sequence of the coat protein of the cowpea strain of tobacco mosaic virus (cowpea virus) has been determined. The tryptic peptide overlaps were obtained by digesting the protein with chymotrypsin and separating and analysing the lysine-and arginine-containing chymotryptic peptides. The primary structure of cowpea virus protein has been found to differ markedly from that of any other known strain of tobacco mosaic virus, and contains 3 amino acid residues more and 96 amino acid changes from the type strain. The significance of the distribution of those areas of the protein in which the amino acid residues are the same for all naturally occurring strains and chemically induced mutants of tobacco mosaic virus so far studied and the residues that form the important carboxyl-carboxylate pairs are discussed.     

Fluorescence studies on the coat protein of alfalfa mosaic virus

The intrinsic luminescence of different forms of the alfalfa mosaic virus (AMV) strain 425 coat protein has been studied, both statically and time resolved. It was found that the emission of the protein (Mr 24,250), which contains two tryptophans at positions 54 and 190 and four tyrosines, is completely dominated by tryptophan fluorescence. The high fluorescence quantum yield indicates that both tryptophans are emitting. Surprisingly, the fluorescence decay is found to be strictly exponential, with a lifetime of 5.1 nsec. Similar results were obtained for various other forms of the protein, i.e. the 30-S polymer, the mildly trypsinized forms of the protein lacking the N-terminal part and the protein assembled into viral particles. Virus particles and proteins of stains S and VRU gave similar results, as well as the VRU protein polymerised into tubular structures. The fluorescence decay is also monoexponential in the presence of various concentrations of the quenching molecules acrylamide and potassium iodide. Stern-Volmer plots were linear and yield for the coat protein dimer with acrylamide a quenching constant of 4.5* 10(8) M-1 sec-1. This indicates that the tryptophans are moderately accessible for acrylamide. For the 30-S polymer a somewhat smaller value was found, whereas in the viral Top a particles the accessibility of the tryptophans is still further reduced. From the decay of the polarisation anisotropy of the fluorescence of the coat protein dimer the rotational correlation time was obtained as 35 nsec. Since this roughly equals the expected rotational correlation time of the dimer as a whole, it suggests that the tryptophans are contained rigidly in the dimer. The results show that in the excited state of the protein the two tryptophans are strongly coupled and suggest that the trp-trp distance is smaller than 10 A. Because the coat protein occurs as a dimer, the coupling can be inter- or intramolecular. The implications for the viral structure are discussed.     

Structural studies on the coat protein of alfalfa mosaic virus. Isolation and characterization of the tryptic peptides and the alignment of the cyanogen-bromide fragments.

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen-bromide cleavage. The tryptic peptides from the protein and its four cyanogen-bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end-group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.     

Primary structure of pilin protein from Bacteroides nodosus strain 216: comparison with the corresponding protein from strain 198

The amino acid sequence of pilin protein from Bacteroides nodosus strain 216 was determined. The protein had a calculated molecular weight of 15962 and contained the same number of amino acid residues (151) as the pilin from the previously sequenced strain 198. The sequence of the first 44 residues was common to both strains, including the unusual amino-terminal amino acid, N-methylphenylalanine. Of the remaining 107 residues, 37% of them differed between the two strains. Comparison of hydrophilicity profiles constructed from the sequence data indicated that a conserved region around residues 71-72 was probably the site of an antigenic determinant.     

Fluorescence studies on the coat protein of Alfalfa Mosaic Virus

Abstract

The intrinsic luminescence of different forms of the alfalfa mosaic virus (AMV) strain 425 coat protein has been studied, both statically and time resolved. It was found that the emission of the protein (M_r24,250), which contains two tryptophans at positions 54 and 190 and four tyrosines, is completely dominated by tryptophan fluorescence. The high fluorescence quantum yield indicates that both tryptophans are emitting. Surprisingly, the fluorescence decay is found to be strictly exponential, with a lifetime of 5.1 nsec. Similar results were obtained for various other forms of the protein, i.e. the 30-S polymer, the mildly trypsinised forms of the protein lacking the N-terminal part and the protein assembled into viral particles. Virus particles and proteins of stains S and VRU gave similar results, as well as the VRU protein polymerised into tubular structures. The fluorescence decay is also monoexponential in the presence of various concentrations of the quenching molecules acrylamide and potassium iodide. Stern-Volmer plots were linear and yield for the coat protein dimer with acrylamide a quenching constant of 4.5 ^* 10⁸ M^?1sec^?1. This indicates that the tryptophans are moderately accessible for acrylamide. For the 30-S polymer a somewhat smaller value was found, whereas in the viral Top a particles the accessibility of the tryptophans is still further reduced. From the decay of the polarisation anisotropy of the fluorescence of the coat protein dimer the rotational correlation time was obtained as 35 nsec. Since this roughly equals the expected rotational correlation time of the dimer as a whole, it suggests that the tryptophans are contained rigidly in the dimer.

The results show that in the excited state of the protein the two tryptophans are strongly coupled and suggest that the trp-trp distance is smaller than 10 A. Because the coat protein occurs as a dimer, the coupling can be inter- or intramolecular. The implications for the viral structure are discussed.     

Viral Coat Protein Peptides with Limited Sequence Homology Bind Similar Domains of Alfalfa Mosaic Virus and Tobacco Streak Virus RNAs

An unusual and distinguishing feature of alfalfa mosaic virus (AMV) and ilarviruses such as tobacco streak virus (TSV) is that the viral coat protein is required to activate the early stages of viral RNA replication, a phenomenon known as genome activation. AMV-TSV coat protein homology is limited; however, they are functionally interchangeable in activating virus replication. For example, TSV coat protein will activate AMV RNA replication and vice versa. Although AMV and TSV coat proteins have little obvious amino acid homology, we recently reported that they share an N-terminal RNA binding consensus sequence (Ansel-McKinney et al., EMBO J. 15:5077–5084, 1996). Here, we biochemically compare the binding of chemically synthesized peptides that include the consensus RNA binding sequence and lysine-rich (AMV) or arginine-rich (TSV) environment to 3′-terminal TSV and AMV RNA fragments. The arginine-rich TSV coat protein peptide binds viral RNA with lower affinity than the lysine-rich AMV coat protein peptides; however, the ribose moieties protected from hydroxyl radical attack by the two different peptides are localized in the same area of the predicted RNA structures. When included in an infectious inoculum, both AMV and TSV 3′-terminal RNA fragments inhibited AMV RNA replication, while variant RNAs unable to bind coat protein did not affect replication significantly. The data suggest that RNA binding and genome activation functions may reside in the consensus RNA binding sequence that is apparently unique to AMV and ilarvirus coat proteins.     

Structural analysis of the coat protein of cucumber green mottle mosaic virus

Using reversed-phase high-performance liquid chromatography, two components of the coat protein of isolate No. 3 of the cucumber green mottle mosaic virus (CGMMV, cucumber strain), Cp1 (minor) and Cp2 (major), were isolated and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In the Cp2 mass spectrum, two polypeptides with Mr of 16,727.0 and 16,813.5 were detected. By Edman degradation in combination with mass spectrometry, the primary structure of the tryptic peptides of Cp2 comprising in total 150 amino acid residues was determined. Two amino acid substitutions, Val-56-->Ala-56 and Asp-64-->Ser-64, were revealed in Cp2, as compared to the watermelon strain of the virus. Cp1 was shown to consist of three polypeptides with Mr of 10,014.2, 10,224.9, and 10,355.9 corresponding to the N-terminal regions of Cp2 (positions 1-92, 1-94, and 1-95). The observed heterogeneity of the coat protein of CGMMV, cucumber strain, may be due to proteolysis during protein isolation.     

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.     

Amino acid sequence of the coat protein subunit in satellite tobacco necrosis virus

The primary structure of the coat protein subunit in satellite tobacco necrosis virus has been investigated. The results obtained are consistent with and support the proposal for the amino acid sequence made from the nucleotide sequence of RNA (Ysebaert et al., 1980). This would imply that no intervening sequences of RNA occur in the cistron for the satellite tobacco necrosis virus coat protein. The polypeptide chain of the protein consists of 195 amino acid residues. It contains one sulfhydryl group but no disulfide bridges. The distribution of various kinds of amino acid residues along the chain is markedly uneven.     

Molecular cloning and nucleotide sequence of the 30K and the coat protein cistron of TMV (tomato strain) genome.

The cDNA copies of tobacco mosaic virus (TMV)-tomato strain (L) genome were cloned by the method of Okayama and Berg (Mol. Cell. Biol. 2, 161-170. (1982)) and the sequence of 1,614 nucleotides at the 3' end was determined. The sequence encompasses the 30K and the coat protein cistron which are located in residues 685-1, 479 and 203-682 from the 3' end of the genome respectively. The close relationship between the tomato and the common strain was shown on the level of the nucleotide sequence. Highly homologous regions are found in the 3' non-coding region, the assembly origin and the 5' flanking region of the 30K protein cistron. The comparison of the deduced amino acid sequence between the tomato and the common strain shows that the 30K protein is composed of the conserved N-terminal four-fifth and the highly divergent region near the C-terminus.     

Enhanced sensitivity to neutralizing antibodies in a variant of equine infectious anemia virus is linked to amino acid substitutions in the surface unit envelope glycoprotein.

Serial passage of the prototype (PR) cell-adapted Wyoming strain of equine infectious anemia virus (EIAV) in fetal donkey dermal (FDD) rather than fetal horse (designated fetal equine kidney [FEK]) cell cultures resulted in the generation of a variant virus strain which produced accelerated cytopathic effects in FDD cells and was 100- to 1,000-fold more sensitive to neutralizing antibodies than its parent. This neutralization-sensitive variant was designated the FDD strain. Although there were differences in glycosylation between the PR and FDD strains, passage of the FDD virus in FEK cells did not reduce its sensitivity to neutralizing antibody. Nucleotide sequencing of the region encoding the surface unit (SU) protein from the FDD strain revealed nine amino acid substitutions compared with the PR strain. Two of these substitutions resulted in changes in the polarity of charge, four caused the introduction of a charged residue, and three had no net change in charge. Nucleotide sequence analysis was extended to the region of the FDD virus genome encoding the extracellular domain of the transmembrane envelope glycoprotein (TM). Unlike the situation with the FDD virus coding region, there were minor variations in nucleotide sequence between individual molecular clones containing this region of the TM gene. Although each clone contained three nucleotide substitutions compared with the PR strain, only one of these was common to all, and this did not affect the amino acid content. Of the remaining two nucleotide substitutions, only one resulted in an amino acid change, and in each case, this change appeared to be conservative. To determine if amino acid substitutions in the SU protein of FDD cell-grown viruses were responsible for the enhanced sensitivity to neutralizing antibodies, chimeric viruses were constructed by using an infectious molecular clone of EIAV. These chimeric viruses contained all of the amino acid substitutions found in the FDD virus strain and were significantly more sensitive to neutralizing antibodies than viruses from the parental (PR) molecular clone. These results demonstrated that sensitivity to neutralizing antibodies in EIAV can be conferred by amino acid residues in the SU protein. However, such amino acid substitutions were not sufficient to enhance cytopathogenicity, as the chimeric viruses did not cause excessive degenererative effects in FDD cells, as was observed with the parental FDD virus strain.     

Primary structure of the potato virus X genome: the region preceding the capsid protein cistron

DNA copies of the potato virus X (PVX) RNA corresponding to 2300 nucleotides at the 3'-end have been cloned. The cloned cDNA copies containing the nucleotides 445-1280 from the 3'-end have been sequenced. The 5'-terminal region of the PVX coat protein gene corresponds to residues 445-786 from the 3'-end. The amino acid sequences of two more open reading frames (ORF) have been deduced from the nucleotide sequence. The potential translation products of these ORF's would correspond to the nonstructural viral proteins. We have located the ORF1 within the region of residues 799-1009 preceding the coat protein cistron. The tentative protein is composed of 70 amino acids and has an aminoterminal segment which is markedly hydrophobic. ORF2 in the PVX sequence ends with UAG at nucleotides 942-944 and extends to the 5'-terminus for additional 340 nucleotides. The distant sequence homology exists between a carboxyterminal portion of PVX ORF2 and that of the nonstructural "30 K-proteins" of the plant tobamoviruses.     

A surface loop of the potato leafroll virus coat protein is involved in virion assembly, systemic movement, and aphid transmission

Two acidic domains of the Potato leafroll virus (PLRV) coat protein, separated by 55 amino acids and predicted to be adjacent surface features on the virion, were the focus of a mutational analysis. Eleven site-directed mutants were generated from a cloned infectious cDNA of PLRV and delivered to plants by Agrobacterium-mediated mechanical inoculation. Alanine substitutions of any of the three amino acids of the sequence EWH (amino acids 170 to 172) or of D177 disrupted the ability of the coat protein to assemble stable particles and the ability of the viral RNA to move systemically in four host plant species. Alanine substitution of E109, D173, or E176 reduced the accumulation of virus in agrobacterium-infiltrated tissues, the efficiency of systemic infection, and the efficiency of aphid transmission relative to wild-type virus, but the mutations did not affect virion stability. A structural model of the PLRV capsid predicted that the amino acids critical for virion assembly were located within a depression at the center of a coat protein trimer. The other amino acids that affected plant infection and/or aphid transmission were predicted to be located around the perimeter of the depression. PLRV virions play key roles in phloem-limited virus movement in plant hosts as well as in transport and persistence in the aphid vectors. These results identified amino acid residues in a surface-oriented loop of the coat protein that are critical for virus assembly and stability, systemic infection of plants, and movement of virus through aphid vectors.