Nucleotide sequences of the 3' leader and 5' trailer regions of human respiratory syncytial virus genomic RNA.

The nucleotide sequences of the 3' extracistronic (leader) and 5' extracistronic (trailer) regions were determined for genomic RNA (vRNA) of human respiratory syncytial virus (RSV) strain A2. To sequence the 3' leader region, vRNA was extracted from purified virions, size-selected, polyadenylated, copied into cDNA, amplified by the polymerase chain reaction, cloned, and sequenced. The 3' leader sequence is 44 nt, which is somewhat shorter than its counterparts (50 to 70 nt) in other nonsegmented negative-strand viruses sequenced to date. The 5' trailer region was mapped and sequenced in part directly by dideoxynucleotide sequencing of vRNA. The sequence was confirmed and completed by analysis of cDNA clones derived from vRNA. The 5' trailer sequence is 155 nt in length, which is substantially longer than its counterparts (40 to 70 nt) in other nonsegmented negative-strand viruses. Ten of the 11 terminal nt of the 3' leader and 5' trailer regions were complementary. Among the other paramyxoviruses, the terminal 5 to 16 nt of the leader and trailer regions are highly conserved, but the corresponding RSV sequences were identical to the others only for the terminal 2 nt of each end. Surprisingly, the termini of the RSV leader and trailer regions were in somewhat better agreement with those of the rhabdoviruses vesicular stomatitis virus and rabies virus, sharing identity for the first 3 or 4 nt.     

Complete nucleotide sequence of Northern cereal mosaic virus and its genome organization

Summary.  We determined the complete nucleotide sequence, 13, 222 nucleotides (nts) of the Northern cereal mosaic virus (NCMV). The genome had 273 nt 5′ trailer sequence and 90 nts 3′leader sequence. It formed a terminal complementarity in 25 nts of both terminal sequences. A characteristic intergenic sequence (consensus) separating genes, 3′-AUUCUUUUUGACUCUAGU-5′ was presented. The genome had nine open reading frames (ORFs) on the viral complementary sequence. Five putative proteins of NCMV were postulated by its molecular weight or comparison of the similarities to other rhabdovirus proteins: nucleocapsid (N), non-structural protein or phosphoprotein (P), matrix protein (M), glycoprotein (G), and polymerase (L). A series of four small ORFs (genes 3 to 6) were also presented between P and M gene sequences. The proposed NCMV genome organization was 3′leader-N-P-3-4-5-6-M-G-L-5′trailer. N and L proteins of NCMV had low but distinct similarities to those of lettuce necrotic yellows virus and Sonchus yellow net virus, respectively. Received September 29, 1999 Accepted January 26, 2000     

Complete genome sequence of rabies virus CVS-24 from China

The entire genome of the mouse-adapted rabies virus strain CVS-24 (challenge virus standard 24), was sequenced. The overall length of the genome was 11,927 nucleotide (nt), comprising a leader sequence of 58 nt, a nucleoprotein (N) gene of 1353 nt, phosphoprotein (P) gene of 894 nt, a matrix protein (M) gene of 609 nt, a glycoprotein (G) gene of 1575 nt, an RNA-dependent RNA polymerase (RdRp, L) gene of 6384 nt and a trailer region of 70 nt. There was a TGAAAAAAA (TG₇) consensus sequence at the end of each gene, except the G gene which had an AGAAAAAAA sequence at the end, and the L/trailer region had the sequence CGAAAAAAA. Three were AACAYYYCT consensus start signals close to TG₇. The five cistrons were separated by intergenic regions (IGRS) of 2, 5, 5, 24 nt, respectively. Residue 333 of the mature G protein, which is considered to be associated with pathogenicity, was Ala in CVS-24. The topology of the phylogenetic trees generated using N protein sequences suggested that CVS-11 and CVS-N₂C have a close relationship to CVS-24.     

Roles of the respiratory syncytial virus trailer region: Effects of mutations on genome production and stress granule formation

The 5′ extragenic trailer region of respiratory syncytial virus (RSV) is known to be necessary for genome replication, but is more than three times the length of the 3′ leader replication promoter, raising the possibility that trailer might play an additional role in viral replication. To examine this, mutant recombinant viruses were constructed in which the trailer region was truncated or substituted with leader-complement sequence. This analysis showed that the complete trailer increased promoter activity, facilitating genome production and viral multiplication. In addition, trailer-containing viruses did not induce stress granules, whereas the leader-complement virus mutant did, resulting in poor multi-cycle viral growth. These data demonstrate that although the RSV trailer does not contain a unique essential sequence, it augments virus growth by enabling optimal genome production. In addition, a sequence at the 5′ terminal end of the trailer region allows RSV to subvert stress granule formation.     

Characterization of human rabies virus vaccine strain in China

Human rabies virus vaccine strain CTN181 from China was sequenced. The overall length of the genome was 11,923 nucleotides (nt), comprising a leader sequence of 58 nt, nucleoprotein (N) gene of 1353 nt, phosphoprotein (P) gene of 894 nt, matrix protein (M) gene of 609 nt, glycoprotein (G) gene of 1575 nt, RNA-dependent RNA polymerase (RdRp, L) gene of 6387 nt, and a trailer region of 70 nt. The five monocistrons are separated by intergenic regions (IGRs) of 2, 5, 5 and 24 nucleotides (nt), respectively. Two obvious differences between CTN181 and the other rabies virus vaccine strains were (1) the putative stop/polyadenylation signal of the G gene has only one poly (A) tract for CTN181, and (2) the start of the open reading frame for L has two repeats of ATG for CTN181. Both were similar to the SHBRV-18 (silver-haired bat-associated RV strain 18) strain. In addition, some mutations and new functional regions were discovered that are presumed crucial to the function of leader region and L protein. There is an equal role for all five genes in the phylogenetics of rabies virus.     

An internal element of the measles virus antigenome promoter modulates replication efficiency

The cis-acting sequence elements that direct measles virus (MV) genome synthesis reside in the 109 base non-coding region at the 5′ trailer (3′ antigenomic) end of MV genome that makes up the antigenomic promoter (AGP). The MV-AGP nucleotides 79–96, corresponding to nucleotide hexamers 14, 15 and 16 (the C′ element), show sequence similarity with the equivalent region of many paramyxoviruses and are analogous to the three nucleotide hexamers that form the second replication control element in the Sendai virus AGP. In this study, results of two independent procedures demonstrate that the MV C′ element also is a replication control sequence. Results of in vivo nucleotide selection experiments show that selection pressure for retaining the wild type nucleotides at the first position of each of the three hexamers, and for the fifth position of the 14th hexamer was relatively high. However, with continued replication, preference for the conservation of wild type nucleotides across the entire C′ element was clearly evident. Results of mutational analysis of individual nucleotides in one or more hexamers in a measles-helper-virus driven reporter gene rescue system agreed with these results. Substitutions at the first position of the 14th, the 15th or the 16th hexamers reduced minireplicon activity dramatically. In contrast, changes at the other five positions of any one hexamer had little or no effect on minireplicon activity, even when all the five bases were changed at the same time. However, when minireplicons were analyzed which contained point mutations at equivalent positions in all three hexamers, it was evident that the nucleotides, particularly those at the 5th position, were also important components of the C′ element. This pattern of sequence requirement in the C′ element based on mutational analysis could be described as a distinct motif, 5′-(GNNNAN)₂GNNNCN-3′, that is important for MV replication.     

Molecular cloning of the NP and L genes of simian virus 5: identification of highly conserved domains in paramyxovirus NP and L proteins.

We have molecularly cloned and determined the nucleotide sequence of the 3' and 5' regions of the genomic RNA of the paramyxovirus simian virus 5 (SV5), including the 3' leader sequence, nucleocapsid protein (NP) gene, large (L) protein gene, and 5' anti-genomic leader (trailer) sequence. The vRNA 3' proximal leader sequence contains 55 nucleotides. The NP gene is 1725 nucleotides in length and encodes a negatively charged protein consisting of 509 residues (MW 56,534). A comparison of the amino acid sequences of 10 paramyxovirus NP proteins indicates a region of high sequence identity near the middle of the protein, and a C-terminal region which is enriched in negatively charged residues. Overall, the SV5 NP protein showed the highest degree of sequence identity with the NP proteins of parainfluenza type 2 virus (58%) and mumps virus (56%). The L gene extends 6804 nucleotides and encodes a positively charged protein consisting of 2255 residues (MW 255,923). The 5' proximal region of the vRNA consists of a 31 nucleotide trailer RNA. The SV5 L protein sequence showed 62% overall identity with the parainfluenza type 2 L protein. Although little overall sequence identity was found between the SV5 and other paramyxovirus L protein sequences, short stretches of extensive amino acid identity were found near the middle of each of the known paramyxovirus L protein sequences, and these common regions may represent sites important for enzymatic activity.     

Nucleotide sequence analysis of Plum pox virus isolate W3174: evidence of a new strain

The nucleotide sequence of Plum pox virus (PPV) isolate W3174 was determined. The virus genome consists of 9788 nucleotides (nt), excluding the poly(A) tail at the 3′-terminus, with 5′- and 3′-untranslated regions (UTRs) of 146 and 219 nt, respectively. The deduced polyprotein consists of 3141 amino acid (aa) residues, with the coat protein region consisting of 993 nt (331 aa). In comparisons with PPV-D, -M, -EA and -C isolates, nucleotide identity levels ranged from 79 to 80% for the entire genome and from 78 to 79%, 78 to 81%, and 92 to 95% for the NIb, CP, and 3′-UTR regions, respectively. The majority of nucleotide substitutions in the NIb region of W3174 are silent, while substitutions in the CP region are not silent, giving aa identities of 89–91% and 79–81%, respectively. The HC-Pro region contains the KITC and PTK motifs, and the DAG motif is located at positions 12–14 of the deduced CP aa sequence, all associated with aphid transmission. Phylogenetic analysis based on the complete genome sequence, the NIb, CP, and 3′-UTR region were performed. PPV-W3174 consistently formed a distinct clade or group, when compared to members of all four recognized strains of PPV, indicating that it is genetically distinct. These results are consistent with serological and nucleic acid-based strain typing data and justify recognition of this isolate as representative of a new strain identified as PPV-W.     

Comparison of the 5' and 3' termini of tomato ringspot virus RNA1 and RNA2: evidence for RNA recombination

The sequences of the 5' terminal 1140 and 3' terminal 1546 nt of tomato ringspot virus (TomRSV) RNA1 have been determined. These sequences share a high degree of nucleotide sequence similarity with the previously determined TomRSV RNA2 sequence. Eighty-eight percent of the 5' terminal 907 nt of TomRSV RNA1 and RNA2 contain identical nucleotide residues; the first 459 nt are identical at all positions, whereas the next 447 nt are identical at only 75.8% of the nucleotide positions. The region of similarity includes not only the 5' nontranslated leader but also sequence probably encoding polyproteins. The 3' terminal 1533 nt of TomRSV RNA1 and RNA2 are identical and are noncoding. The sequences common to RNA1 and RNA2 account for almost 35% of the total genomic sequence. It is possible that the similar sequences at both ends of TomRSV RNA1 and RNA2 are a result of recombination between these two genomic RNA components.     

Determination of the complete genomic sequence and analysis of the gene products of the virus of Spring Viremia of Carp, a fish rhabdovirus

The complete genome of spring viremia of carp virus (SVCV) was cloned and the sequence of 11019 nucleotides was determined. It contains five open reading frames (ORF's) encoding for the nucleoprotein N; phosphoprotein P; matrix protein M; glycoprotein G; and the viral RNA dependent RNA polymerase L. Genes are organised in the order typical for rhabdoviruses: 3'-N-P-M-G-L-5'. The short leader and trailer regions of SVCV exhibit inverse complementarity and are similar to the respective 3' and 5' ends of the genome of vesicular stomatitis virus. To verify the predicted open reading frames proteins were expressed in bacteria and analysed with a polyclonal anti-SVCV serum. Furthermore, monospecific antisera against the distinct viral proteins were generated. Comparison of genome and protein confirm the assignment of SVCV to the genus Vesiculovirus.     

Sequence of the 3' untranslated region of brome mosaic virus coat protein messenger RNA

The 3' terminal 337 bases of BMV (brome mosaic virus) coat protein mRNA (BMV RNA4) are presented. This sequence includes the terminal portion of the coat protein cistron and the complete 300-base 3' noncoding sequence. The 3' noncoding sequence displays significant complementarity to the 5' terminal sequence of BMV RNA3 but not to the 5' terminal sequence of BMV RNA4.     

Intergeneric relationship between the Aspergillus ochraceous virus F and the Penicillium stoloniferum virus S

It was reported that the ‘slow’ component (PsV-S) of Penicillium stoloniferum virus complex also occurred in a second genus, Aspergillus ochraceous. The responsible virus for this intergeneric occurrence was considered to be the ‘fast’ component (AoV-F) of A. ochraceous virus complex. In this investigation, AoV dsRNA 1, that was previously shown to cross-hybridize with PsV-S dsRNA, has been cloned. It was 1754 bp in length and contained one open reading frame of 539 amino acids (p63), had the same genome organization as PsV-S dsRNA S1 and also had the conserved sequence motif of the PsV-S dsRNAs (5′-GCGCAAAA-3′) at the 5′ terminus. A BLAST search indicated that p63 was a putative dsRNA-dependent RNA polymerase (RdRp), had 81% of sequence homology to members of the genus Partitivirus, and grouped together with PsV-S in phylogenetic analysis. But immunoblot analysis showed that the capsid protein (P3) of AoV-F virus component did not reacted against PsV-S antiserum. These evidences suggest that the cross serological relationship between AoV-F and PsV-S previously observed may have been due to the RdRps of the respective viruses rather than between their respective capsid proteins as was assumed in 1985.     

The fourth genus in the Orthomyxoviridae: sequence analyses of two Thogoto virus polymerase proteins and comparison with influenza viruses

The tick-borne Thogoto virus (THOV) is the type species of a newly recognized fourth genus, Thogotovirus, in the family Orthomyxoviridae. Because of the distant relationship of THOV with the influenza viruses, determination of its genomic information can potentially be used to identify important domains in influenza virus proteins. We have determined the complete nucleotide sequence of the second longest RNA segment of THOV. The molecule comprises 2212 nucleotides with a single large open reading frame encoding a protein of 710 amino acids, estimated M_r 81 284. The protein shares 77% amino acid similarity with the PB1-like protein of Dhori virus, a related tick-borne virus, and 50–53% with the PB1 polymerase proteins of influenza virus A, B and C. All the motifs characteristic of RNA-dependent polymerases were identified including the SSDD motif common to all RNA-dependant RNA polymerases, indicating that the THOV protein is functionally analogous to the influenza virus PB1 proteins and involved in chain elongation. We also report the corrected sequence of the third longest RNA segment of THOV, encoding a protein which shares 44–47% amino acid similarity with the PA-like polymerase proteins of influenza virus A, B and C. The biological significance of conserved domains in these orthomyxovirid proteins is discussed.     

Malakal virus from Africa and Kimberley virus from Australia are geographic variants of a widely distributed ephemerovirus

Kimberley virus (KIMV) is an arthropod-borne rhabdovirus that was isolated in 1973 and on several subsequent occasions from healthy cattle, mosquitoes (Culex annulirostris) and biting midges (Culicoides brevitarsis) in Australia. Malakal virus (MALV) is an antigenically related rhabdovirus isolated in 1963 from mosquitoes (Mansonia uniformis) in Sudan. We report here the complete genome sequences of KIMV (15442 nt) and MALV (15444 nt). The genomes have a similar organisation (3′-l-N-P-M-G-G_NS-α1-α2-β-γ-L-t-5′) to that of bovine ephemeral fever virus (BEFV). High levels of amino acid identity in each gene, similar gene expression profiles, clustering in phylogenetic analyses of the N, P, G and L proteins, and strong cross-neutralisation indicate that KIMV and MALV are geographic variants of the same ephemerovirus that, like BEFV, occurs in Africa, Asia and Australia.     

The partial purified RNA-dependent RNA polymerases from bamboo mosaic potexvirus and potato virus X infected plants containing the template-dependent activities

RNA-dependent RNA polymerases (RdRp) isolated from bamboo mosaic potexvirus (BaMV) and potato virus X infected Nicotiana benthamiana plants and solubilized with the detergent NP-40, generated a full-length genomic and two subgenomic double-stranded RNAs of respective viruses in an in vitro RdRp assay containing endogenous RNA templates. Template-dependent and species-specific RdRp activity could be detected after the removal of endogenous RNA templates. The 3′ untranslated regions (UTR) containing a stretch of 40 adenylate residues were shown to be an efficient exogenous RNA template for in vitro RdRp reactions. Solution hybridization and nuclease digestion studies revealed that the products transcribed in vitro were minus-sense. Besides using the 3′ UTR for minus-sense RNA synthesis, the BaMV RdRp can also recognize 3′ terminal 77 nucleotides of the minus-strand for plus-sense RNA synthesis. Promoter studies with BaMV RdRp showed that domain D containing the potexviral hexamer motif of the 3′ UTR would be the major contributor of minus-sense RNA synthesis in vitro. On the other hand, the pseudoknot domain containing the poly(A) sequences would be sufficient for minus-sense RNA synthesis.     

Specific interactions of HeLa cell proteins with Coxsackievirus B3 RNA: La autoantigen binds differentially to multiple sites within the 5' untranslated region

Translation initiation of the coxsackievirus B3 (CVB3) RNA occurs by internal ribosomal entry. The internal ribosomal entry site (IRES) of the virus has been mapped to the 5′ untranslated region (5′ UTR) of the genome. As well, the 5′ UTR has been suggested to play roles in determining the tissue tropism and infectivity of the virus. In this study, we investigated interactions between HeLa cell protein extracts and radiolabeled RNA of CVB3 5′ UTR by competitive UV cross-linking. We have observed a number of proteins that specifically interact with the three sub-cloned regions of the 5′ UTR. In particular, the molecular weights of five of these proteins resemble those of the eukaryotic translation initiation factors 4A, 4B and 4G, as well as the La autoantigen and the polypyrimidine tract binding protein. Based on this data, we focused on the interaction of the 5′ UTR with the La autoantigen, which was purified by the glutathione-S-transferase affinity method. We have confirmed the highly specific interaction of the La autoantigen with the 5′ UTR sequence nt 210–529. The core IRES (nt 530–630) and nt 1–209 also appear to bind to the La protein at moderate and weak affinities, respectively. A functional role of the La autoantigen in translation initiation is suggested.     

Nucleotide sequence of the 5′-terminus of Newcastle disease virus and assembly of the complete genomic sequence: agreement with the “rule of six”

Summary.  We have determined the sequences of the 5′ ends of three strains of Newcastle disease virus, permitting the assembly of the entire genomic sequence, which amounts to 15,186 nucleotides. This length is in agreement with the rule of six, which has been shown to determine replication efficiency in similar viruses. Comparison of the extreme 5′ end of the trailer sequence with that of the 3′-terminal leader sequence of the virus reveals a high degree of complementarity. Variation between the 5′-terminal sequences of the different strains reveals the presence of alternative L gene polyadenylation signals, leading to correspondingly different trailer lengths. Received March 17, 1998 Accepted May 31, 1998     

Comparison of the full-length genome sequence of avian metapneumovirus subtype C with other paramyxoviruses

We determined the nucleotide (nt) sequence of the small hydrophobic (SH), attachment glycoprotein (G), and RNA polymerase (L) genes, plus the leader and trailer regions of the Colorado strain of Avian metapneumovirus subtype C (aMPV/C) in order to complete the genome sequencing. The complete genome comprised of 13,134 nucleotides, with a 40 nt leader at its 3' end and a 45 nt trailer at its 5' end. The aMPV/C L gene was the largest with 6173 nt and consisting of a single open reading frame encoding a 2005 amino acids (aa) protein. Comparison of the aMPV/C SH, G, and L nt and predicted aa sequences with those of Human metapneumoviruses (hMPV) revealed higher nt and aa sequence identities than the sequence identities between the aMPV subtypes A, B, C, and D, supporting earlier finding that aMPV/C was closer evolutionary to hMPV than the other aMPV subtypes.