Sequencing and characterization of the coat protein and 3′ non-coding region of a newsweet potato potyvirus

Summary.  A cDNA library was constructed from viral genomic RNA purified from sweet potato plants affected by “Sweet Potato Chlorotic Dwarf disease” in an attempt to clarify the etiology of this viral complex in Argentina. By sequence analysis, some of the obtained clones were found to belong to sweet potato feathery mottle potyvirus (SPFMV), to a closterovirus and to a new potyvirus. A cDNA clone of 1,103 bp representing the coat protein cistron and 3^′ non-coding region of the newly identified potyvirus was further characterized. The sequence contained an ORF of 855 nucleotides with a coding capacity of 285 amino acids, followed by a 3^′ untranslated tail of 248 nucleotides. The core and C-terminal regions have sequences well conserved among potyviruses. Furthermore, amino acid sequence comparisons of the capsid protein with those of other described potyviruses showed 63% homology with SPFMV, 68 to 70% with two different isolates of sweet potato latent potyvirus (SPLV), 57% with sweet potato G potyvirus (SPGV) and 73% with potato virus Y (PVY). These data allowed us to propose the inclusion of this virus as a new member of the family Potyviridae, genus Potyvirus with the designation sweet potato mild speckling potyvirus (SPMSV). Received January 16, 1997 Accepted March 4, 1997     

Complete nucleotide sequence and genome organization of sweet potato feathery mottle virus (S strain) genomic RNA:the large coding region of the P1 gene

Summary. The complete nucleotide sequence of a sweet potato feathery mottle virus severe strain (SPFMV-S) genomic RNA was determined from overlapping cDNA clones and by directly sequencing viral RNA. The viral RNA genome is 10 820 nucleotides long, excluding the poly(A) tail and contains one open reading frame (ORF) starting at nucleotide 118 and ending at 10 599, potentially encoding a polyprotein of 3 493 amino acids (Mr 393 800). The ORF was followed by a 3 untranslated region of 221 nucleotides. The deduced polyprotein includes P1 (74K), HC-Pro (52K), P3 (46K), 6K1, CI (72K), 6K2, NIa-VPg (22K), NIa-Pro (28K), NIb (60K) and coat (35K) proteins, after an analysis of protein cleavage sites analogous to other potyvirus polyproteins. The polyprotein had a high level of amino acid identity with those of other potyviruses, except in the regions of P1 and P3. The P1 of SPFMV-S RNA has 664 amino acid residues, and is the largest and least similar to those of other potyviruses. HC-Pro and CI show high identity with those of other potyviruses. P3 has relatively low identity, however, the length of P3 was within the range of variability among other potyviruses. The 6K1 protein between P3 and C1 is also highly similar to those of other potyviruses. This is the first report on the complete nucleotide sequence of the sweet potato-infecting virus. Received October 28, 1996 Accepted April 3, 1997     

Molecular characterization and coat protein serology of watermelon leaf mottle virus (Potyvirus)

Summary.  A cDNA library was generated from purified RNA of watermelon leaf mottle virus (WLMV) (Genus Potyvirus). Two overlapping clones totaling 2,316 nucleotides at the 3′terminus of the virus were identified by immunoscreening with coat protein antiserum. The sequence analyses of the clones indicated an open reading frame (ORF) of 2,050 nucleotides which encoded part of the replicase and the coat protein, a 243-nucleotide non-coding region (3′UTR), and 23 adenine residues of the poly (A) tail. The taxonomic status of WLMV was determined by comparisons of the sequence of the cloned coat protein gene and 3′UTR with potyvirus sequences obtained from GenBank. The nucleotide sequence identities of WLMV compared with 17 other potyviruses ranged from 55.6 to 63.5% for the coat protein, and from 37.2 to 48.3% for the 3′UTR. Phylogenetic analyses of the coat protein region and the 3′UTR indicated that WLMV did not cluster with other potyviruses in a clade with high bootstrap support. The coat protein gene was expressed in Escherichia coli and a polyclonal antiserum was prepared to the expressed coat protein. In immunodiffusion tests, WLMV was found to be serologically distinct from papaya ringspot virus type W, watermelon mosaic virus 2, zucchini yellow mosaic virus, and Moroccan watermelon mosaic virus. In Western blots and ELISA, serological cross-reactivity with other cucurbit potyviruses was observed. Serological and sequence comparisons indicated that watermelon leaf mottle virus is a distinct member of the Potyvirus genus. Accepted September 23, 1999     

Cloning and nucleotide sequence of the capsid protein and the nuclear inclusion protein (NIb) of potato virus A

Summary The sequence of the 3-terminal 2597 nucleotides of potato virus A (PVA) genome has been determined from cDNA clones. An open reading frame was identified and potentially encodes a large polyprotein containing 789 amino acid residues. This large open reading frame was found to have a high similarity to the nuclear inclusion protein (NIb) and the capsid protein (CP) genes of several potyviruses. The data suggest that the PVA NIb and CP are products arising from the maturation of the large polyprotein as observed for other potyviruses. Putative cleavage sites corresponded to consensus sequences NIa/NIb and NIb/CP, respectively, of other potyviruses. The NIb (putative RNA polymerase) and CP are expected to be 516 and 269 amino acid residues (M_r of 58,939 and 30,094), respectively. The non-coding region is 227 nucleotides long, rich in A and U and unlike other viruses. Furthermore, there are two AUG codons in frame in front of the capsid protein gene suggesting an alternative mode for the capsid protein expression.     

Phylogenetic relationships of closely related potyviruses infecting sweet potato determined by genomic characterization of Sweet potato virus G and Sweet potato virus 2

Complete nucleotide sequences of Sweet potato virus G (SPVG) and Sweet potato virus 2 (SPV2) were determined to be 10,800 and 10,731 nucleotides, respectively, excluding the 3'-poly(A) tail. Their genomic organizations are typical of potyviruses, encoding a polyprotein which is likely cleaved into 10 mature proteins by three viral proteinases. Conserved motifs of orthologous proteins of viruses in the genus Potyvirus are found in corresponding positions of both viruses. Pairwise comparisons of individual protein sequences of the two viruses with those of 78 other potyviruses show that P1 protein and coat protein (CP) of both viruses are significantly large, with the SPVG CP as the largest among the all the known species of the genus Potyvirus. The extended N-terminal region of the P1 protein is conserved in the potyviruses and ipomovirus infecting sweet potato. A novel ORF, PISPO, is identified within the P1 region of SPVG, SPV2, Sweet potato feathery mottle virus (SPFMV), and Sweet potato virus C (SPVC). The C-terminal half of CP is highly conserved among SPFMV, SPVC, SPVG, SPV2, and Sweet potato virus-Zimbabwe. Phylogenetic analysis based on the deduced CP amino acid sequences supports the view that these five viruses are grouped together in a SPFMV lineage. The analysis also reveals that Sweet potato virus Y and Ipomoea vein mosaic virus are grouped with SPV2 as one species, and these two viruses should be consolidated with SPV2.     

Characterization of petunia flower mottle virus (PetFMV), a new potyvirus infecting Petunia x hybrida

Summary.  With the introduction of cutting-grown Petunia x hybrida plants on the European market, a new potyvirus which showed no serological reaction with antisera against any other potyviruses infecting petunias was discovered. Infected leaves contained flexuous rod-shaped virus particles of 750 – 800 nm in length and inclusion bodies (pinwheel structures) typical for potyviruses in ultrathin leaf sections. The purified coat protein with a M_r of approximately 36 kDa could be detected in Western immunoblots with a specific antibody to the coat protein of the petunia-infecting virus. The 3′ end of the viral genome encompassing the 3′ non-coding region, the coat protein gene, and part of the NIb gene was amplified from infected leaf material by IC/PCR using degenerate and specific primers. Sequences of PCR-generated cDNA clones were compared to other known sequences of potyviruses. Maximum homology of 56% was found in the 3′ non-coding region between the petunia isolate and other potyviruses. A maximum homology of 69% was found between the amino acid sequence of the coat protein of the petunia isolate and corresponding sequences of other potyviruses. These data indicate that the petunia-infecting virus is a previously undescribed potyvirus and the name petunia flower mottle virus (PetFMV) is suggested. Accepted November 5, 1997 Received July 25, 1997     

Sequence of full length cDNA for human S-adenosylhomocysteine hydrolase.

Two cDNA clones for human S-adenosylhomocysteine hydrolase isolated from a placental cDNA library were sequenced. Each contained a sequence of 1299 nucleotides encoding a 432 amino-acide protein of MW 47660. Clone 16-1 contained 47 nucleotides 5' of the coding region, and a 780 nucleotide 3' flanking region terminating in apoly A tail. In addition, a 101 nucleotide unprocessed intron interrupted the coding sequence at nucleotide 854 (second base of codon 285). Clone 20-1 contianed 43 nucleotides 5' and 742 nucleotides 3' flanking the uninterrupted coding region. Besides the intron, the clones differed in one position of the coding sequence and at two positions of the 3' non-coding region. The cDNAs for human and rat S-adenosylhomocysteine hydrolase were identical at 91.5% of position in the coding sequence and showed 70% homology in the 3' non-coding regions. Human and rat S-adenosylhomocysteine hydrolases are identical at 97% of amino-acid residues, and the Dictyostelium and human enzymes at 75%.     

Complete nucleotide sequence of the mRNA coding for the N protein of vesicular stomatitis virus (New Jersey serotype)

The nucleotide sequence of the mRNA encoding the nucleocapsid protein of the New Jersey serotype (Ogden strain) of vesicular stomatitis virus (VSV) was determined from two overlapping cDNA clones spanning almost entirely the coding region of the mRNA. The 5'-terminal noncoding sequence present in the mRNA but not in the cDNA clones was determined from a primer extended to the 5' terminus of the mRNA. The mRNA is 1329 nucleotides long (excluding polyadenylic acid) and encodes a protein of 422 amino acids. The nucleotide sequence was compared with the previously determined nucleotide sequence of the nucleocapsid protein of the Indiana serotype. An overall identity of 67.7% was found between the two serotypes. The only place where insertions and/or deletions have occurred during the evolution of the two viral genes from their presumed common ancestor is in the untranslated region. The nonidentical nucleotides are distributed throughout the length of the mRNA although not in an entirely random manner. The predicted amino acid sequence demonstrates that both proteins are initiated from the initiator codon located at the same distance from the 5' end (nucleotides 14 to 16) and contain the same number of amino acids. An overall identity of more than 80% of the amino acid sequence was observed between the two proteins when conservative replacements of amino acids were considered.     

Nucleotide sequence of the capsid protein gene of potato virus Y (PVY)

The nucleotide sequence of the 3 terminal region of potato virus Y (PVY) was determined. Starting with a poly(A) tail of 18 residues a non-coding region of 335 nucleotides precedes the region encoding for the virus coat protein (cp) 801 nucleotides long ending with a TGA. This region was located by comparing the predicted amino acid sequence with the one determined for the PVY capsid protein by Shukla et al. (1). Both sequences contained 267 amino acids sharing about 94% homology. They differ, however, at several positions presumably due to base transitions within their respective nucleotide sequences. Restriction endonuclease sites in and around the cp coding region were identified.     

Molecular taxonomy of a new potyvirus isolated from chilli pepper in Thailand

Summary.  A virus isolated from chilli pepper plants in Kamphaengsaen, Nakorn Pathom, showing vein banding mottle symptoms was classified using sequence analysis and phylogeny of the coat protein gene and 3′ noncoding region (3′ NCR). This virus was found to be a typical potyvirus on the basis of particle morphology, biological properties and cytopathology. The 3′ terminus region of the genome of 1,309 nucleotides, representing the viral coat protein gene and 3′ NCR was cloned and sequenced. Nucleotide sequence analysis indicated that the 3′ region of the viral genome had a polyA tail of at least 12 nucleotides, a noncoding region of 272 nucleotides, a coat protein gene of 864 nucleotides and 161 nucleotides representing the 3′ terminus of the polymerase gene. The amino acid sequence of the coat protein was compared with those of 23 distinct potyviruses, and 63.1% shown to be the highest homology. However the 3′ NCR had, at most, 29.7% random homology, thus indicating that this virus is a distinct species in the genus Potyvirus in the family Potyviridae. The result is well supported by previous studies on the biology and biochemical properties of this virus. Received February 13, 1998 Accepted June 26, 1998     

Complete nucleotide sequence of the matrix protein mRNA of mumps virus

The complete nucleotide sequence of the mumps virus membrane protein or matrix protein (M) has been determined by sequencing cDNA clones and confirmed by partially sequencing the M mRNA and the genome. The mRNA is 1248 nucleotides long excluding the poly(A) and encodes a protein of 375 amino acids. The molecular weight (38,670), deduced from the amino acid sequence, is in agreement with the molecular weight of the viral M protein estimated by polyacrylamide gel electrophoresis (39-40 kDa). The mumps virus M protein shows 23-27% homology with M proteins of Newcastle disease virus (NDV), measles virus, canine distemper virus (CDV), parainfluenza virus type 3, and Sendai virus, respectively. A comparison of the M protein sequences of the above six paramyxoviruses did not reveal any conserved area of homology common among all paramyxovirus M proteins.     

Selection of polyclonal antibodies to the N terminus of bean yellow mosaic potyvirus coat protein by induction of tolerance with monoclonal antibody

Polyclonal antisera to potyviruses contain virus-specific as well as cross-reacting antibodies. The virus-specific antibodies are directed to the surface-located, N-terminal region of the coat protein, whereas cross-reacting antibodies are produced against multiple epitopes within the core region of the coat protein (minus N and C termini), which displays extensive sequence homology among distinct potyviruses. In the present study, immunological tolerance was induced in mice against the cross-reactive central core region of bean yellow mosaic virus (BYMV) using a rat monoclonal antibody (mAb) to the L3T4 molecule (the mouse equivalent of CD4). Generation of specific antisera reactive to the N terminus of BYMV was attained in tolerized mice by secondary immunization with whole viral coat protein from BYMV. This approach appears to be ideally suited to potyviruses where a two-third of the coat protein molecule contains immunogenic epitopes which can result in cross-reacting antibodies.     

Nucleotide sequence of the coat protein coding region of the potyvirus tobacco vein-banding mosaic virus

Summary The sequence of the 3 1184 nucleotides of tobacco vein-banding mosaic virus (TVBMV) genome has been determined. It contains a single open reading frame which encompasses the whole of the coat protein of TVBMV. The sequence of the first 20 amino acids at the N-terminal region of the coat protein has also been determined chemically to be GDDQTVDAGKNVQSNQKQRN. The sequence matches the translation product of the open reading frame starting with amino acid-271; a glycine residue. Thus the coat protein of TVBMV has a calculated M_r of 30 210. The 3 non-coding region of TVBMV is 185 nucleotides in length. Sequence alignment of the coat proteins or the 3 non-coding regions from TVBMV and other reported potyviruses indicated that TVBMV is a separate species of the potyvirus genus.     

Nucleotide sequence analysis of the coat protein genes of two Korean isolates of sweet potato feathery mottle potyvirus

Summary.  The coat protein (CP) genes of the genomic RNA of two Korean isolates of sweet potato feathery mottle potyvirus (SPFMV), SPFMV-K1 and SPFMV-K2, were cloned and their complete nucleotide sequences were determined. Sequence comparisons of the two Korean isolates showed 97.8% amino acid identity in the CP cistron, and 79.9% to 99.0% identity with those of 6 other known SPFMV strains. Of 74 amino acid changes totally among the SPFMV strains, 39 changes were located at the N-terminal region. Pairwise amino acid sequence comparison revealed sequence similarities of 48.6 to 70.2% between SPFMV and 20 other potyviruses, indicating SPFMV to be a quite distinct species. Multiple alignment of the CP cistrons from other potyviruses showed that most of the conserved amino acid residues of the genus Potyvirus are well preserved in the corresponding locations. Accepted November 13, 1997 Received September 1, 1997     

Biochemical analysis of the capsid protein gene and capsid protein of tobacco etch virus: N-terminal amino acids are located on the virion's surface

The sequence of the 1491 nucleotides found at the 3' end of the genome of the highly aphid-transmissible (HAT) isolate of tobacco etch virus (TEV) has been determined. The nucleotide sequence of the capsid protein gene has been identified and compared with the corresponding region of the not-aphid-transmissible (NAT) isolate of TEV and with pepper mottle virus (PeMV). The deduced amino acid sequences of the two TEV capsid proteins displayed 98% homology and a 66% homology with PeMV capsid protein. Three of the six amino acid differences between the capsid proteins of the two TEV isolates occurred near the N terminus of the protein. Biochemical and immunological evidence suggested the N-terminal 29 amino acids of the capsid protein were hydrophilic and were located at or near the virion's surface.     

cDNA clones coding for the complete murine B chain of complement C1q: nucleotide and derived amino acid sequences

We have isolated cDNA clones covering the complete B chain of the complement subunit C1q from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide, all of the 228 amino acids of the mature protein and 140 nucleotides of the 3' untranslated region, including a poly A addition signal. The coding region for the mature protein contains 261 nucleotides for the Gly-X-Y repeat and 408 nucleotides for the globular portion of C1q. By comparing the nucleotide sequence of the mouse B chain of C1q with the B chain from human (Reid, K. B. M., 1985, Biochem. J. 231, 729), we find a high homology (80%) within the mature protein, a lower homology within the signal peptide (59%) and the 3' untranslated region (47%) and no homology (26%) in the 5' untranslated region.     

The complete genome sequence of pepper severe mosaic virus and comparison with other potyviruses

Summary. The complete nucleotide sequence of pepper severe mosaic virus (PepSMV) was determined. The viral genome consisted of 9890 nucleotides, excluding a poly (A) tract at the 3′ end of the genome. The PepSMV RNA genome encoded a single polyprotein of 3085 amino acid residues, resulting in ten functionally distinct potyviral proteins. The lengths of the 5′ nontranslated region (NTR) and the 3′ NTR were 164 and 468 nucleotides, respectively. The genome organization of the virus was typical for members of the genus Potyvirus in the family Potyviridae. The coat protein amino acid sequence identity between PepSMV and the other 45 potyviruses ranged from 53.4 to 79.7%. Sequence alignments and phylogenetic analyses of the potyviral polyprotein sequences revealed that PepSMV was the closest to potato virus Y (PVY) and closely related to members of the PVY subgroup. Our genome sequence data clearly confirmed that PepSMV belongs to a separate species in the genus Potyvirus.     

cDNA clones coding for the complete murine B chain of complement Clq: nucleotide and derived amino acid sequences

We have isolated cDNA clones covering the complete B chain of the complement subunit Clq from mouse; this subunit initiates the classical complement pathway. Deoxynucleotide sequence analysis shows that these clones contain 156 nucleotides of the 5' untranslated region, followed by sequences coding for the 25 amino acids of the signal peptide; all of the 228 amino acids of the mature protein; and 140 nucleotides of the 3' untranslated region, including a poly A additional signal. The coding region for the mature protein contains 261 nucleotides for the Gly-X-Y repeat and 408 nucleotides for the globular portion of Clq. By comparing the nucleotide sequence of the mouse B chain of Clq with the human B chain (Reid, K.B.M., 1985, Biochem. J. 231, 729), we find a high homology (80%) within the mature protein, a lower homology within the signal peptide (59%) and the 3' untranslated region (47%) and no homology (26%) in the 5' untranslated region.