Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins with those of other flaviviruses

We have determined the complete sequence of the RNA of dengue 2 virus (S1 candidate vaccine strain derived from the PR-159 isolate) with the exception of about 15 nucleotides at the 5' end. The genome organization is the same as that deduced earlier for other flaviviruses and the amino acid sequences of the encoded dengue 2 proteins show striking homology to those of other flaviviruses. The overall amino acid sequence similarity between dengue 2 and yellow fever virus is 44.7%, whereas that between dengue 2 and West Nile virus is 50.7%. These viruses represent three different serological subgroups of mosquito-borne flaviviruses. Comparison of the amino acid sequences shows that amino acid sequence homology is not uniformly distributed among the proteins; highest homology is found in some domains of nonstructural protein NS5 and lowest homology in the hydrophobic polypeptides ns2a and 2b. In general the structural proteins are less well conserved than the nonstructural proteins. Hydrophobicity profiles, however, are remarkably similar throughout the translated region. Comparison of the dengue 2 PR-159 sequence to partial sequence data from dengue 4 and another strain of dengue 2 virus reveals amino acid sequence homologies of about 64 and 96%, respectively, in the structural protein region. Thus as a general rule for flaviviruses examined to date, members of different serological subgroups demonstrate 50% or less amino acid sequence homology, members of the same subgroup average 65-75% homology, and strains of the same virus demonstrate greater than 95% amino acid sequence similarity.     

Partial nucleotide sequence of the Japanese encephalitis virus genome

Approximately 10 kb of the estimated 10.9-kb genome of the Japanese encephalitis virus (JE; Nakayama strain) has been cloned as cDNA; the uncloned portion includes 430 bases at the 5'-terminus and 450 bases at the 3'-end. A map of the genome has been developed through nucleotide sequencing and in vivo expression with the Escherichia coli expression vector lambda gt11 and immunological identification. Sequence results for 4320 nucleotides suggest the JE genome organization is very similar to those of three other flaviviruses for which sequence information is available. Like the other flaviviruses, the JE proteins are encoded by a single open reading frame that continues uninterrupted throughout the region sequenced. Considerable homology exists between the JE RNA and protein sequences and those of the other characterized flaviviruses. Comparative nucleotide and (amino acid) homology values for the M-E-NS1-ns2 segment of JE are approximately MVE, 70% (80%), WN, 68% (76%), and YF, 50% (45%). Even greater homology is suggested when the protein hydrophobicity profiles are compared. The molecular relationships are consistent with the established serological relationships among JE, MVE, and WN viruses and argue that these flaviviruses may have been derived from a common evolutionary ancestor.     

Sequence analysis of the membrane protein V3 of the flavivirus West Nile virus and of its gene

Flaviviruses contain a large membrane-associated protein V3, having a mol mass of about 50 kDa which is responsible for hemagglutination. We have isolated the V3 protein from the West Nile (WN) flavivirus and determined its amino-terminal amino acid sequence and amino acid sequences of fragments derived from this protein. We have also transcribed parts of the WN virus genome RNA into cDNA and cloned and sequenced this cDNA. The results of these analyses have allowed us to identify the region of the viral genome coding for the V3 protein. In this report we describe the total nucleotide sequence of the genome region coding for the WN virus V3 protein and the amino acid sequence of the V3 protein derived from these analyses. The exact carboxy terminus of the V3 protein has not been determined in these experiments. These analyses have shown that the V3 protein of WN virus does not contain an Asn-X-Ser/Thr sequence which could allow addition of N-linked carbohydrate chains to this protein. In accordance with this finding, analyses of metabolic labeling of the V3 protein using [3H]glucosamine indicate that the WN virus V3 protein is an unglycosylated protein. Together with our earlier analyses these results show that the viral structural proteins are present on the genome RNA in the order 5'-terminus-core protein (V2)-small membrane-associated protein (NV2)-large membrane-associated protein (V3) and describe the nucleotide sequences coding for all WN virus structural proteins identified so far. A hypothesis concerning the processes involved in the synthesis of all viral structural proteins and the probable orientation of these proteins relative to the endoplasmatic reticulum membrane based on the structure of these proteins is discussed.     

Primary structure of the West Nile flavivirus genome region coding for all nonstructural proteins

The genome RNA of the flavivirus West Nile (WN) virus has been transcribed into cDNA, the cDNA has been cloned, and the nucleotide sequences coding for the structural proteins have been determined (Castle et al., 1985; Wengler et al., 1985). We have now determined the nucleotide sequence coding for all viral nonstructural proteins which comprises 7929 nucleotides. Together with our earlier sequence analyses these data show that a long open reading frame (ORF) containing 10,290 nucleotides is present on the genome of WN virus. The two largest nonstructural proteins which can be detected in flavivirus-infected cells are the proteins NV5 and NV4 which have an apparent molecular mass of 97,000 and 74,000 Da, respectively. Both proteins were isolated by preparative polyacrylamide gel electrophoresis, and partial amino acid sequences of peptides derived from these proteins were determined. These analyses allow us to localize the nucleotide regions which code for these proteins and show that the region coding for the NV5 protein is located at the 3'-terminus of the long ORF. Together with our earlier analyses these data show that the protein sequences of virus-specific proteins are present on the viral polyprotein translated from the long ORF in the order V2-NV2-V3-(nonstructural proteins of up to 75,000 Da)-NV4-(nonstructural proteins of up to 45,000 Da)-NV5. Our data indicate that virus-specific structural and nonstructural proteins which are synthesized from a single long ORF accumulate in large amounts in infected cells. A possible role of the presence of these molecules, which are associated to cellular membranes, in the accumulation of membrane vesicles which characteristically occurs in flavivirus-infected cells is discussed.     

Nucleotide sequence and deduced amino acid sequence of the structural proteins of dengue type 2 virus, Jamaica genotype

The nucleotide sequence of the 5'-terminal 2469 bases of dengue 2 (Jamaica genotype) virus has been determined and the encoded proteins compared with those of yellow fever and West Nile viruses, which belong to different flavivirus serogroups. The cDNA clone which was sequenced contains a 5'-noncoding region of 96 nucleotides followed by a single open reading frame coding for the structural proteins 5'-C-prM(M)-E-3' and the beginning of the NS1 nonstructural protein. The amino acid sequence homology between the structural polyprotein precursor of dengue 2 virus and those of yellow fever and West Nile viruses is 36.5 and 42%, respectively. The dengue virus structural proteins are similar in size and composition to those of the other flaviviruses. The basic capsid protein and the membrane and envelope proteins have hydrophobic regions at their C termini. The dengue 2 capsid C, membrane M, and envelope E proteins share 13, 36, and 43% homology, respectively, with the cognate proteins of yellow fever virus, and 33, 32, and 47% homology with the cognate proteins of West Nile virus. All 6 cysteine residues in the dengue 2 premembrane protein and all 12 cysteine residues in the dengue 2 envelope protein are conserved in the cognate proteins of yellow fever and West Nile viruses.     

Processing of dengue virus type 2 structural proteins containing deletions in hydrophobic domains

Summary The 5 end of the genome of the dengue virus type 2 encoding the structural proteins was expressed using recombinant vaccinia virus. Three additional recombinants derived by deletion of selected dengue sequences within the parental construct were also expressed. They were designed to assess the role of hydrophobic domains in the processing of the viral polyprotein in intact cells. The first construct contained a deletion of nucleotides encoding most of the C protein; nucleotides encoding the hydrophobic domain at the carboxy terminus were retained. The second and third constructs contained smaller deletions of 72 bp and 129 bp encoding hydrophobic domains at the carboxy termini of C and prM respectively. Indirect immunofluorescence and radioimmunoprecipitation were used to detect prM and E in cells infected with recombinant viruses. The results showed that deletion of 90% of C had no apparent effect on the processing of prM and E, and that the signal sequence for E at the carboxy terminus of prM was active in the absence of the upstream signal sequence for prM at the carboxy terminus of C. Deletion of the hydrophobic sequences preceding the amino terminus of E prevented cleavage at the prM-E junction. These results obtained using infected cells were consistent with the published findings for the translation of flavivirus RNA in vitro, and indicated the importance of membrane association in the cleavage of structural proteins from the flavivirus polyprotein. In addition, cells infected with the recombinant virus containing the large deletion in the C coding region released the E glycoprotein into the culture medium.     

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.     

Terminal sequences of the genome and replicatioe-form RNA of the flavivirus west nile virus: absence of poly(A) and possible role in RNA replication

The structures of the infectious 42 S genome RNA of the flavivirus West Nile (WN) virus and of the replicative-form (RF) RNA containing 42 S RNA of positive and negative polarity have been investigated. The RF RNA has been labeled in vitro at the 3′ and 5′ termini and the terminal sequences have been determined by the mobility shift method. The results obtained indicate that both RNA molecules are exact complements of each other and that the 3′ terminus of the 42 S plus-strand RNA component of the RF RNA does not contain a poly(A) sequence but terminates with a heteropolymeric AACACAGGAUCU_OH sequence. The 3′ terminus of the 42 S minus-strand RNA has the sequence CUCACACAGGCGAACUACU_OH. Comparison of these sequences shows that both molecules contain the 3′-terminal dinucleotide CUOH and the heptanucleotide ACACAGG which is separated from the 3′-terminal dinucleotide by two and seven nucleotides in 42 S plus- and minus-strand RNA, respectively. The 42 S viral genome RNA also does not contain a 3′-terminal poly(A) sequence but terminates with the 3′-terminal sequence identified in the 42 S plus-strand RNA of the RF. Analysis of the nucleotides adjacent to the cap at the 5′ terminus of the viral genome RNA together with the 3′-terminal sequence analysis indicates that the nucleotide sequence of the viral genome RNA is identical to that of the 42 S plus-strand RNA component of the virus-specific RF RNA.     

Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of dengue type 2 virus, Jamaica genotype: comparative analysis of the full-length genome

The sequence of the 5'-end of the genome of dengue 2 (Jamaica genotype) virus has been previously reported (V. Deubel, R. M. Kinney, and D. W. Trent, 1986, Virology 155, 365-377). We have now cloned and sequenced the remaining 75% of the genomic RNA that encodes the nonstructural proteins. The complete genome is 10,723 bases in length with a single open reading frame extending from nucleotides 97 to 10,269 encoding 3391 amino acids. The 3'-noncoding extremity presents a stem- and loop-structure and contains a repeated oligonucleotide sequence. Comparisons of the nucleotide sequences of the genomes of dengue 2 viruses of different topotypes reveal 90-95% similarity, with 64-66% similarity evident between dengue viruses of different serotypes. The amino acid sequence of the polyprotein of dengue 2 Jamaica virus shows 97, 68, 50, and 44% similarity with those of other dengue 2, dengue 1, or dengue 4, West Nile, and yellow fever viruses, respectively. Despite amino acid sequence divergence, the hydrophobic profile of the flavivirus proteins is highly conserved. Proteins NS1, NS3, and NS5 are the most conserved. Conserved amino acid stretches present in all flavivirus proteins may be involved in common essential biological functions.     

Nucleotide sequence of the 26 S mRNA of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and deduced sequence of the encoded structural proteins

A cDNA clone containing all of the 26 S mRNA coding region of the RNA genome of Venezuelan equine encephalitis (VEE) virus, virulent strain Trinidad donkey (TRD), has been constructed and sequenced. The nucleotide and deduced amino acid sequences of the 26 S RNA of VEE virus conform to the general organization of the alphavirus subgenomic mRNA. Excluding the poly(A) tail, the VEE 26 S RNA is 3913 nucleotides long with a protein coding region of 3762 nucleotides. Codon usage in the translated region is nonrandom and correlates well with that reported for Sindbis (SIN), Semliki Forest (SF), and Ross River (RR) alphaviruses. Highly conserved sequences of 19 to 22 nucleotides representing putative replicase recognition sites occur at the 26 S RNA junction region of the 42 S genomic RNA and at the 3′ terminus immediately preceding the poly(A) tail. The conserved sequence at the 26 S/42 S junction region of VEE virus differs from that of other alpha-viruses in that an ochre termination codon (UAA) is substituted for a GGU (Gly) codon present in the other viruses. The 5′ and 3′ noncoding regions (30 and 121 nucleotides, respectively) of the VEE 26 S RNA are shorter than has been reported for several other alphaviruses. The approximate transmembrane domains of the VEE E1 and E2 envelope glycoproteins have been identified. VEE E1 contains a single asparagine-linked glycosylation site, whereas E2 has three such sites, all of which are apparently glycosylated. The deduced amino acid sequence of the VEE polyprotein shows an overall homology of 44 to 46% with the precursor polyproteins of SIN, SF, and RR viruses. VEE virus capsid, E1, and E2 structural proteins show 43 to 46%,50 to 53%,and 36 to 41% homology, respectively, with the cognate proteins of SIN, SF, and RR viruses.     

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.     

Sequence and organization of the genomic termini of equine herpesvirus type 1

The nucleotide sequence and organization of the genomic termini and of the junction of the long (L) and short (S) regions of the equine herpesvirus type 1 genome were determined. Sequencing of the XbaI-Q fragment (1441 nucleotides) revealed that the left terminus contains sets of inverted repeat and direct repeat sequences. The terminal sequence is described as DR1-UC-DR4 (18, 60, and 16 nucleotides, respectively) because of its homology to these elements of the 'a' sequence of herpes simplex virus. Located at each terminus of the S region as part of the inverted repeats is a 54 nucleotide sequence with homology to the Ub element of the HSV 'a' sequence. Thus, these data suggest that fusion of the EHV-1 genomic termini during replication will generate a sequence equivalent to Ub-DR1-Uc-DR4, which is known to be an ideal cleavage/packaging signal in herpesviral DNAs. Eighty-seven nucleotides of the L region left terminus sequence are repeated in an inverted fashion at nucleotide 892; also a 32 basepair portion, DR1-Uc (18 and 14 basepairs respectively), is reiterated 20 times in an inverted fashion as part of a 54 basepair tandem repeat located at the other L region terminus (L-S junction). It is not known whether these small inverted repeats at the L termini mediate isomerization of the L region at a very low level. The organization of the terminal sequences of the EHV-1 genome and the similarity of these sequences to the cleavage/packaging elements of other herpesviruses are discussed.     

Genome sequences of a mouse-avirulent and a mouse-virulent strain of Ross River virus

The nucleotide sequence of the genomic RNA of a mouse-avirulent strain of Ross River virus, RRV NB5092 (isolated in 1969), has been determined and the corresponding sequence for the prototype mouse-virulent strain, RRV T48 (isolated in 1959), has been completed. The RRV NB5092 genome is approximately 11,674 nucleotides in length, compared with 11,853 nucleotides for RRV T48. RRV NB5092 and RRV T48 have the same genome organization. For both viruses an untranslated region of 80 nucleotides at the 5' end of the genome is followed by a 7440-nucleotide open reading frame which is interrupted after 5586 nucleotides by a single opal termination codon. By homology with other alphaviruses, the 5586-nucleotide open reading frame encodes the nonstructural proteins nsP1, nsP2, and nsP3; a fourth nonstructural protein, nsP4, is produced by read-through of the opal codon. The RRV nonstructural proteins show strong homology with the corresponding proteins of Sindbis virus and Semliki Forest virus in terms of size, net charge, and hydropathy characteristics. However, homology is not uniform between or within the proteins; nsP1, nsP2, and nsP4 contain extended domains which are highly conserved between alphaviruses, while the C-terminal region of nsP3 shows little conservation in sequence or length between alphaviruses. An untranslated "junction" region of 44 nucleotides (for RRV NB5092) or 47 nucleotides (for RRV T48) separates the nonstructural and structural protein coding regions. The structural proteins (capsid-E3-E2-6K-E1) are translated from an open reading frame of 3762 nucleotides which is followed by a 3'-untranslated region of approximately 348 nucleotides (for RRV NB5092) or 524 nucleotides (for RRV T48). Excluding deletions and insertions, the genomes of RRV NB5092 and RRV T48 differ at 284 nucleotides, representing a sequence divergence of 2.38%. Sequence deletions or insertions were found only in the noncoding regions and include a 173-nucleotide deletion in the 3'-untranslated region of RRV NB5092, compared with RRV T48. In the coding regions, most of the nucleotide differences are silent; there are 36 amino acid differences in the nonstructural proteins and 12 in the structural proteins. The distribution of amino acid differences between the two RRV strains correlates with the location of domains which are poorly conserved in sequence between alphaviruses. The possible role of amino acid differences in envelope glycoproteins E1 and E2 in determining the different antigenic and biological properties of RRV NB5092 and RRV T48 is discussed.     

The viral RNA 3′- and 5′-end structure and mRNA transcription of infectious salmon anaemia virus resemble those of influenza viruses

The nucleotide sequences of the termini of two of the genomic segments of the negative strand RNA virus infectious salmon anaemia virus (ISAV) were determined. The sequence of the terminal 9 nucleotides at both ends of the viral RNAs was identical, and showed distinctive sequence homology with the conserved terminal sequences found in the orthomyxoviruses. For both ISAV genomic segments a computer-based secondary structure modelling indicated that the terminal 21-24 nucleotides were able to form self-complementary panhandle structures. Comparison with ISAV-derived mRNA sequences showed that ISAV mRNAs have heterogeneous 5'-ends, and are polyadenylated from a signal sequence 13-14 nucleotides downstream of the 5'-end terminus of the vRNA. Furthermore, the in vitro replication of ISAV was hindered by the RNA polymerase II inhibitor alpha-amanitin. These findings indicate that the mechanisms for replication of ISAV are similar to those of the orthomyxoviruses, and add to the previously reported structural similarities between ISAV and the orthomyxoviruses.     

Nucleotide sequence of dengue type 3 virus genomic RNA encoding viral structural proteins

Complementary DNAs to the 5 proximal region of the dengue virus type 3 RNA were cloned into bacterial plasmids and the nucleotide sequence of 3,000 bases from the 5 terminus of the genome were determined by DNA and RNA sequencing methods using dideoxy chain-termination reactions. Comparison of the nucleotide sequence thus obtained with those of other flavivirus genomes revealed significant homology existing in nucleotide sequence of the flavivirus genomes. When we compared amino acid sequence deduced from the nucleotide sequence with those of other flaviviruses, this genome region was found to include sequences encoding three viral structural proteins C, M, and E and a part of the viral nonstructural protein NS1 in this order in addition to the 5-noncoding sequence. The characteristics and functions of these proteins were discussed based on the deduced amino acid sequences and their hydrophobic profiles. The genetic relationship of flaviviruses was also discussed based on the genetic variation observed in their genomes.