Dugbe nairovirus M RNA: Nucleotide sequence and coding strategy

The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated. The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA). Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments. No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products. Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies. Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein. Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1. The G2 coding region was located upstream of the G1 sequence. The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.     

Complete nucleotide sequence of the M RNA segment of Rift Valley fever virus

The entire M RNA segment of the phlebovirus Rift Valley fever virus (RVFV) has been molecularly cloned and the complete nucleotide sequence determined. The RNA is 3884 nucleotides in length, corresponding to a molecular weight of 1.38 X 10(6), having a base composition of 27.3% A, 25.4% G, 27.2% U, and 20.1% C. Sequences present at the 3' and 5' termini of the molecule are largely complementary for some 51 residues and can form a stable duplex structure when the potential secondary structure of the entire molecule is considered. A single major open reading frame, capable of encoding 1206 amino acids (131,845 Da), was found in the viral-complementary sequence ("positive" polarity). Amino-terminal amino acid sequencing of the purified viral glycoproteins G1 and G2 allowed for the positioning of the coding sequences for these polypeptides within this major open reading frame in the following orientation with respect to the genomic M RNA: 3'-G2-G1-5'. From the predicted amino acid composition of the two mature viral glycoproteins, both were found to have a high cysteine content (G2, 6%; G1, 5%). Sequences within the open reading frame capable of encoding up to 23,000 Da of polypeptide were found in addition to those required for the viral glycoproteins. The potential contribution of these sequences to the coding capacity of the M RNA, viral protein processing, and intracellular protein distribution is discussed.     

The nucleotide sequence of Indian peanut clump virus RNA 2: sequence comparisons among pecluviruses

Summary.  The RNA-2 molecule of an isolate of the L serotype of Indian peanut clump virus (IPCV) was shown to consist of 4290 nucleotides with five open reading frames (ORF). The arrangement of the ORFs resembled that in RNA-2 of Peanut clump virus (PCV) from West Africa. The proteins encoded by the ORFs in IPCV-L RNA are between 32% and 93% identical to those encoded by PCV RNA. Partial sequence data for the RNA-2 of isolates of the H and T serotypes of IPCV show that the coat and P40 proteins encoded by the 5′-most ORFs of RNA-2 of IPCV-L, IPCV-H and IPCV-T are as similar to each other as any is to the corresponding proteins of PCV. A conserved motif ‘F-E-x₆-W’ is present near the C-termini of the coat proteins of all three IPCV serotypes and of PCV, as it is in the coat proteins of other viruses that have rod-shaped particles, such as Tobacco mosaic virus and Tobacco rattle virus. The results support the distinction of IPCV and PCV as separate virus species, but also raise the question of how the serotypes of IPCV should be classified. Received December 17, 1999/Accepted March 15, 2000     

Complete nucleotide sequence of the Japanese encephalitis virus genome RNA

The complete nucleotide sequence of the Japanese encephalitis virus (JEV) genome RNA was determined. The JEV genome contains 10,976 nucleotides and encodes a single long open reading frame (ORF) of 10,296 nucleotides corresponding to 3432 amino acid residues. This long polypeptide is thought to be cleaved into three structural proteins and several nonstructural proteins of the virus. The genetic location of the three structural proteins was determined by comparing the deduced amino acid sequence from the nucleotide sequence with the N-terminal amino acid sequences that were determined from the three purified structural proteins. The C-terminal region of the ORF may encode a RNA-dependent RNA polymerase which has significant sequence homology with those of other RNA viruses.     

Complete nucleotide sequence of dengue type 3 virus genome RNA

The complete nucleotide sequence of the genome of the dengue virus type 3 was determined. Sequence analyses of the genomic RNA and cloned cDNA revealed that the genomic RNA contains 10,696 nucleotides and encodes a single open reading frame of 10,170 nucleotides corresponding to 3390 amino acid residues. The N-terminal amino acid sequences of three structural proteins (C, M, and E proteins) and the preM protein were also determined from the purified virion. When the deduced amino acid sequence and N-terminal amino acid sequence determined from purified proteins were compared with those of other flaviviruses, the genome organization was found to be the same as that of other flaviviruses.     

Complete nucleotide sequence of the genomic RNA of Sindbis virus

The entire nucleotide sequence of the genomic RNA of the type virus of the alphavirus genus, Sindbis virus, has been determined. The genome is 11,703 nucleotides in length, exclusive of the 5' cap and the 3'-terminal poly(A) tract. After the 5'-terminal cap there are 59 nucleotides of 5' nontranslated nucleic acid followed by a reading frame of 7539 nucleotides that encodes the nonstructural polypeptides and which is open except for a single opal termination codon. Following 48 untranslated bases located in the junction region which separates the nonstructural and structural protein coding sequences, there is an open reading frame 3735 nucleotides long that encodes the structural proteins. Finally, the 3' untranslated region is 322 nucleotides long. The nonstructural proteins are translated from the genomic RNA as two polyprotein precursors. The first is 1896 amino acids in length and terminates at an opal codon at position 1897. This polyprotein is processed to produce three polypeptides called nsP1, nsP2, and nsP3. Sites of post-translational cleavage to produce these three proteins have been tentatively located using available N-terminal amino acid sequence data. In both cases cleavage probably occurs between the two alanine residues in the sequence Gly-Ala-Ala. The fourth nonstructural protein, nsP4, is produced when readthrough of the opal codon produces a second polyprotein precursor of length 2513 amino acids, which is also cleaved posttranslationally. The structural proteins are translated from a subgenomic message which begins at nucleotide 7598, is 4106 nucleotides in length (exclusive of the poly(A) tract), and is coterminal with the 3' end of the genomic RNA. The structural proteins are also translated as a polyprotein precursor which is cleaved to produce a nucleocapsid protein and two integral membrane glycoproteins as well as two small peptides not present in the mature virion. A replication strategy for Sindbis virus based upon the complete nucleotide sequence, as well as prior data, is presented.     

Complete nucleotide sequence of Radish mosaic virus RNA polymerase gene and phylogenetic relationships in the genus Comovirus

The 3'-terminal part of RNA1 genome segment of Radish mosaic virus (RaMV) including complete RNA polymerase gene was sequenced. The 207 amino acids long polymerase is matured from a polyprotein precursor by cleavage at putative Q/H site by viral protease. The alignment of available amino acid sequences of RNA polymerase genes of comoviruses revealed a closest (55%) identity of RaMV to Red clover mottle virus (RCMV).     

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 cloning and complete nucleotide sequence of galinsoga mosaic virus genomic RNA

Summary.  The complete genomic sequence of galinsoga mosaic virus (GaMV) was determined. The genome consists of 3 803 nucleotides and has five open reading frames (ORFs). The 5′ ORF (ORF 1) encodes a protein with predicted molecular mass of 23 kDa and readthrough of its amber stop codon probably yields a 82 kDa protein (ORF 2). ORFs 3 and 4 encode two polypeptides with molecular masses of 8 and 7 kDa, respectively. ORF 5 encodes the 36 kDa capsid protein. Amino acid sequence comparisons revealed that the nonstructural proteins encoded by ORFs 1, 3, and 4 were more similar to the corresponding gene products of tobacco necrosis virus, strain A, than to those of carmoviruses. Conversely, the coat protein was more similar to that of tombusviruses. The readthrough region of the viral replicase (ORF 2) had high sequence homology with that of carmo-, tombus-, and necroviruses. Computer analysis of the protein encoded by ORF 1 as well as of the corresponding product of turnip crinkle (TCV) and melon necrotic spot (MNSV) carmoviruses revealed the presence of a sequence with local hydrophobicity and hydrophobic moment characteristic of mitochondrial targeting sequence which may explain the origin of the carmovirus-induced multivesicular bodies from mitochondria. Accepted August 25, 1997 Received June 18, 1997     

The nucleotide sequence of the coding region of tobacco etch virus genomic RNA: evidence for the synthesis of a single polyprotein

The complete nucleotide sequence of the tobacco etch virus (TEV) RNA genome has been determined excepting only the nucleotide(s) present at the extreme 5' terminus. The assembled TEV genomic sequence is 9496 nucleotides in length followed by a polyadenylated tract ranging from 20 to 140 residues. A computer search of the sequence reveals the following. A 5' untranslated region, rich in adenosine and uridine, is present between nucleotides 1 and 144. A putative initiation codon, at nucleotides 145-147, marks the beginning of a large open-reading frame (ORF) which ends with an opal (UGA) termination codon at positions 9307-9309. A 186-nucleotide untranslated region is present between the termination codon of the ORF and the beginning of the 3' polyadenylated region. The predicted translation product of this ORF is a 3054 amino acid polyprotein with a mol wt of 345,943. A function for the large (54,000 Mr) nuclear inclusion protein is suggested by a comparison of the deduced amino acid sequence with a protein data bank. This protein displays biochemical similarities to other viral RNA-dependent, RNA polymerases.     

Complete nucleotide sequence of four RNA segments of fig mosaic virus

The complete sequence of four viral RNA segments of fig mosaic virus (FMV) was determined. Each of the four RNAs comprises a single open reading frame (ORF) 7,093, 2,252, 1,490 and 1,472 nucleotides in size, respectively. These ORFs encode the following proteins in the order: RNA-dependent RNA polymerase (p1 264 kDa), a putative glycoprotein (p2 73 kDa), a putative nucleocapsid protein (p3 35 kDa) and a protein with unknown function (p4 40.5 kDa). All RNA segments possess untranslated regions containing at the 5′ and 3′ termini a 13-nt complementary sequence. A conserved motif denoted premotif A was found to be present in addition to the five RdRp motifs A–F in RNA-1. In phylogenetic trees constructed with the amino acid sequences of RNA-1 and RNA-2, FMV clustered consistently with European mountain ash ringspot-associated virus (EMARaV) in a clade close to those comprising members of the genera Hantavirus, Orthobunyavirus and Tospovirus. The amino acid sequence of the putative FMV nucleocapsid protein encoded by RNA-3 shared identity with comparable sequences of EMARaV and the unclassified viruses pigeonpea sterility mosaic virus (PPSMV) and maize red stripe virus (MRSV). The nucleocapsid sequences rooted the four viruses in a clade close to the genus Tospovirus. Based on molecular, morphological and epidemiological features, FMV appears to be very closely related to PPSMV and MRSV. All these viruses are phylogenetically related to EMARaV and therefore seem to be eligible for classification in the proposed genus Emaravirus, which, in turn, may find a taxonomic allocation in the family Bunyaviridae.     

The complete nucleotide sequence of turnip mosaic virus RNA Japanese strain

Summary The complete nucleotide sequence of the RNA genome of turnip mosaic virus Japanese strain (TuMV-J) has been determined from five overlapping cDNA clones and by direct sequencing of viral RNA. The RNA sequence was 9833 nucleotides in length, excluding a 3 terminal poly(A) tail. An AUG triplet at position 130–132 was assigned as the initiation codon for the translation of the genome size viral polyprotein which would consist of 3164 amino acid residues. Interestingly, a different amino acid sequence (continuous twenty amino acids) within the cytoplasmic inclusion protein between TuMV-J and Canadian strain of TuMV was observed, caused by an insertion and a deletion of nucleotides.DDBJ/EMBL/GenBank accession number D83184.     

Ross River virus 26 s RNA: complete nucleotide sequence and deduced sequence of the encoded structural proteins

The complete sequence of the 26 S RNA of Ross River virus (T48 strain) has been obtained and from this the amino acid sequences of the encoded structural proteins have been deduced. These include a basic capsid protein and two envelope glycoproteins. The nucleotide sequence was obtained by chemical sequence analysis of both single-stranded and double-stranded cDNA made to RNA and the sequence data so obtained was rapidly aligned by making use of the protein homology found among the alphaviruses. The polyprotein precursor encoded by the 26 S RNA of Ross River virus is 75% homologous to that of Semliki Forest virus and 48% homologous to that of Sindbis virus. The extent of homology is not uniform within a protein or between proteins and this is discussed with respect to the possible function of the various polypeptide domains in the virus life cycle. In each case the putative attachment site of the amino proximal carbohydrate chains of the three glycoproteins is conserved, whereas the attachment site of a second chain, if present, is not conserved. The 3'-untranslated region of Ross River virus RNA is 524 nucleotides long. It contains a sequence of about 50 nucleotides in length which is present in four copies but which is not shared with other alphaviruses examined.     

Complete nucleotide sequence of the duck plague virus gE gene

Archives of Virology -     

Complete nucleotide sequence of the M RNA segment of Uukuniemi virus encoding the membrane glycoproteins G1 and G2

We have determined the complete nucleotide sequence of the virion M RNA segment of Uukuniemi virus (Uukuvirus genus, Bunyaviridae) from cloned cDNA. The RNA that encodes the two membrane glycoproteins G1 and G2 is 3231 residues long (mol wt 1.1 X 10(6)). The 5' and 3' ends of the RNA are partially complementary to each other for some 30 bp, enabling the formation of a stable panhandle structure (delta G = -40 kcal/mol) and the circularization of the molecule. The extreme 5' and 3' terminal nucleotides are identical for 10 to 13 residues to those of the M RNA of Punta Toro and Rift Valley fever viruses, two members of the Phlebovirus genus. A single open reading frame comprising 1008 amino acid residues (mol wt 113,588) was found in the mRNA-sense strand between nucleotides 18 and 3042. This probably corresponds to the previously identified 110,000-Da precursor (p110) of G1 and G2. By comparing the partial aminoterminal sequences of purified G1 and G2 with the deduced protein sequence we confirmed that the gene order is NH2-G1-G2-COOH. Both mature G1 and G2 are preceded by a stretch of 17 predominantly hydrophobic amino acids likely to represent the signal sequences. At their COOH-terminal ends, G1 and G2 have a hydrophobic stretch of amino acids, 19 and 27 residues, respectively, that probably anchors the proteins to the lipid bilayer. The sequence indicates that mature G2 is 495 amino acids long (mol wt 54,869), whereas the exact size of G1 is unclear, since the location of the COOH-terminus of G1 is not known. An upper value of 479 amino acids (mol wt 55,181) can, however, be suggested. Both G1 and G2 contain four potential glycosylation sites for Asn-linked glycans and both are unusually rich in cysteines, 6.1% in G1 and 5.4% in G2. Comparison of the amino acid sequence of the M RNA product of Uukuniemi virus with that of Punta Toro and Rift Valley fever viruses showed in both cases a weak homology that was more pronounced for the proteins located at the COOH-terminal end of the precursor. This suggests a distant evolutionary relationship between the Phlebo- and Uukuvirus genera.     

The primary nucleotide sequence of the bovine leukemia virus RNA packaging signal can influence efficient RNA packaging and virus replication

Two RNA stem-loop structures in the gag gene have been implicated as representing the primary encapsidation (packaging) signal for bovine leukemia virus (BLV), a member of the Delta retrovirus of the Retroviridae. In this study, we conducted an analysis of these RNA structures, stem loop 1 (SL1) and stem loop 2 (SL2), to determine if both the loop and the stem nucleotide bases are important for RNA encapsidation. We have found that the primary sequence of the unpaired bases located in the loop regions of both SL1 and SL2 are important for efficient RNA encapsidation and virus replication. The primary sequence of the bases that form the stems for both SL1 and SL2 was observed to aid in efficient encapsidation and replication. We also observed that the order of SL1 and SL2 is important for RNA encapsidation and virus replication efficiency. A viral RNA with two copies of either SL1 or SL2 was found to replicate and package RNA as efficiently as a viral RNA with only one copy of SL1 or SL2. This provides evidence that SL1 and SL2 are not functionally equivalent. Sequences from human T cell leukemia virus type 1 (HTLV-1) that are located in the same region of HTLV-1 as the SL1 and SL2 of BLV were used to replace the BLV SL1, SL2, or both in a BLV RNA. These BLV RNAs were still encapsidated and replicated, suggesting that these sequences may function as an encapsidation signal in HTLV-1. The chimeric RNAs did not replicate as well as the parental, indicating that the primary nucleotide sequence along with the secondary and tertiary structure of the RNA plays a role in efficient RNA encapsidation and replication.     

Evolutionary relationships in the ilarviruses: nucleotide sequence of prunus necrotic ringspot virus RNA 3

Summary.  The complete nucleotide sequence of an isolate of prunus necrotic ringspot virus (PNRSV) RNA 3 has been determined. Elucidation of the amino acid sequence of the proteins encoded by the two large open reading frames (ORFs) allowed us to carry out comparative and phylogenetic studies on the movement (MP) and coat (CP) proteins in the ilarvirus group. Amino acid sequence comparison of the MP revealed a highly conserved basic sequence motif with an amphipathic α-helical structure preceding the conserved motif of the ‘30K superfamily’ proposed by Mushegian and Koonin [26] for MP’s. Within this ‘30K’ motif a strictly conserved transmembrane domain is present in all ilarviruses sequenced so far. At the amino-terminal end, prune dwarf virus (PDV) has an extension not present in other ilarviruses but which is observed in all bromo- and cucumoviruses, suggesting a common ancestor or a recombinational event in the Bromoviridae family. Examination of the N-terminus of the CP’s of all ilarviruses revealed a highly basic region, part of which resembles the Arg-rich motif that has been characterized in the RNA-binding protein family. This motif has also been found in the other members of the Bromoviridae family, suggesting its involvement in a structural function. Furthermore this region is required for infectivity in ilarviruses. The similarities found in this Arg-rich motif are discussed in terms of this process known as genome activation. Finally, phylogenetic analysis of both the MP and CP proteins revealed a higher relationship of A1MV to PNRSV, apple mosaic virus (ApMV) and PDV than any other member of the ilarvirus group. In that sense, A1MV should be considered as a true ilarvirus instead of forming a distinct group of viruses. Received June 4, 1996 September 25, 1996