Nucleotide Sequence Of RNA Segment Research Articles

Phylogenetic analysis of influenza C virus nonstructural (NS) protein genes and identification of the NS2 protein.

The nucleotide sequences of RNA segment 7 (nonstructural protein gene; NS) were compared among 34 influenza C virus strains isolated between 1947 and 1992. The results showed that all the NS genes analysed had the potential to encode NS1 and NS2 proteins of 246 and 182 amino acids, respectively. The deduced amino acid sequence of the previously unidentified NS2 was fairly well conserved, although it was more divergent than the NS1 protein sequence. Moreover, immunoprecipitation experiments with rabbit immune serum against a glutathione S-transferase fusion protein containing the C-terminal region of the 182 amino acid NS2 protein revealed synthesis of a protein with an apparent molecular mass of approximately 22 kDa in infected cells. A phylogenetic analysis showed that the 34 NS genes were split into two distinct groups, A and B. Comparison of the phylogenetic positions of the individual isolates in the NS gene tree with those in the haemagglutinin-esterase (HE) gene tree suggested that most of the influenza C viruses currently circulating in Japan, irrespective of their HE gene lineage, had acquired group B NS genes through reassortment events that presumably occurred either in the 1970s or in the early 1980s.

Molecular characterization of double-stranded RNA segments encoding the major capsid proteins of a Palyam serogroup orbivirus that caused an epizootic of congenital abnormalities in cattle.

cDNA cloning of the double-stranded RNA genome of Chuzan virus, a member of the Palyam serogroup orbiviruses, was carried out and the complete nucleotide sequences of RNA segments 2, 3, 6 and 7, encoding the major capsid proteins VP2, VP3, VP5 and VP7, respectively, were determined. The individual segments had single open reading frames and short inverted repeats adjacent to the conserved terminal sequences. Comparative sequence analysis with other serogroups of the genus Orbivirus suggested that VP2 is the principal determinant of serotype specificity and the neutralizing antigen of the Palyam serogroup. VP5 is also considered to be associated with antigenic variability. Both VP3 and VP7 probably contain serogroup-specific epitopes. Phylogenetic profiles demonstrated that the Palyam serogroup virus is more closely related to African horsesickness virus than to bluetongue virus and epizootic haemorrhagic disease virus.

Comparison of the non-structural protein, NS1, of tick-borne and insect-borne orbiviruses

The nucleotide sequence of RNA segment 6 of Broadhaven virus (BRDV), a tick-borne orbivirus, was determined principally from two overlapping cDNA clones and RNA end sequence analysis. The genome segment is 1714 base pairs in length and has a coding capacity for a protein of 537 amino acids, having a net charge of +4.0 at neutral pH. Comparison of the predicted amino acid sequence of BRDV RNA segment 6 with the NS1 sequence of insect-borne orbiviruses, bluetongue virus (BTV), African horse sickness virus (AHSV) and epizootic haemorrhagic disease virus (EHDV) of deer, revealed amino acid identities of 21, 22 and 21%, respectively. This compares with amino acid identities of 31 to 50% between the NS1 proteins of these gnat-transmitted orbiviruses. A recombinant baculovirus was produced containing a full-length clone of BRDV segment 6, which expressed a protein of 61 kD in infected Spodoptera frugiperda cells. Like other orbivirus NS1 proteins the expressed protein formed tubules similar to those produced in BRDV-infected BHK21 cells.

Complete nucleotide sequence of RNA segment 3 of bluetongue virus serotype 2 (Ona-A). Phylogenetic analyses reveal the probable origin and relationship with other orbiviruses

The nucleotide sequence of the RNA segment 3 of bluetongue virus (BTV) serotype 2 (Ona-A) from North America was determined to be 2772 nucleotides containing a single large open reading frame of 2703 nucleotides (901 amino acid). The predicted VP3 protein exhibited general physiochemical properties (including hydropathy profiles) which were very similar to those previously deduced for other BTV VP3 proteins. Partial genome segment 3 sequences, obtained by polymerase chain reaction (PCR) sequencing, of BTV isolates from the Caribbean were compared to those from North America, South Africa, India, Indonesia, Malaysia and Australia, as well as other orbiviruses, to determine the phylogenetic relationships amongst them. Three major BTV topotypes (Gould, A.R. (1987) Virus Res. 7, 169–183) were observed which had nucleotide sequences that differed by approximately 20%. At the molecular level, geographic separation had resulted in significant divergence in the BTV genome segment 3 sequences, consistent with the evolution of distinct viral populations. The close phylogenetic relationship between the BTV serotype 2 (Ona-A strain) from Florida and the BTV serotypes 1, 6 and 12 from Jamaica and Honduras, indicated that the presence of BTV serotype 2 in North America was probably due to an exotic incursion from the caribbean region as previously proposed by Sellers and Maaroof ((1989) Can. J. Vet. Res. 53, 100–102) based on trajectory analysis. Conversely, nucleotide sequence analysis of Caribbean BTV serotype 17 isolates suggested they arose from incusions which originated in the USA, possibly from a BTV population distinct from those circulating in Wyoming.

Identification of a second protein encoded by influenza C virus RNA segment 6.

Influenza C virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 6. Unspliced mRNA from this RNA segment, which has not been previously identified, can potentially encode a polypeptide that contains an additional 132 amino acids on the carboxy terminus of the M1 protein. Here the nucleotide sequences of RNA segment 6 of four influenza C strains, isolated in Japan between 1964 and 1988, were compared with the previously determined sequence of C/Ann Arbor/1/50. The results indicated that the deduced amino acid sequence of the carboxy-terminal 132 amino acid domain is conserved fairly well although it is more divergent than the M1 protein sequence. Examination of RNA segment 6-specific mRNAs also showed that unspliced mRNA is present, although in small quantities (approximately 13% of spliced mRNA), in influenza C virus-infected cells. To search for a polypeptide encoded by the unspliced mRNA, the extra carboxy-terminal domain was expressed in Escherichia coli as the glutathione S-transferase fusion protein, and rabbit immune serum was raised against the purified fusion protein. Immunoprecipitation experiments with this antiserum revealed that a previously unrecognized protein of apparent M(r) approximately 18,000, designated CM2, is synthesized in influenza C virus-infected cells.

Growth restriction of influenza A virus by M2 protein antibody is genetically linked to the M1 protein.

The M2 protein of influenza A virus is a 97-amino acid integral membrane protein expressed at the surface of infected cells. Recent studies have shown that a monoclonal antibody (14C2) recognizes the N terminus of M2 and restricts the replication of certain influenza A viruses. To investigate the mechanism of M2 antibody growth restriction, 14C2 antibody-resistant variants of strain A/Udorn/72 have been isolated. Most of the variant viruses are not conventional antigenic variants as their M2 protein is still recognized by the 14C2 antibody. A genetic analysis of reassortant influenza viruses prepared from the 14C2 antibody-resistant variants and an antibody-sensitive parent virus indicates that M2 antibody growth restriction is linked to RNA segment 7, which encodes both the membrane protein (M1) and the M2 integral membrane protein. Nucleotide sequence analysis of RNA segment 7 from the variant viruses predicts single amino acid substitutions in the cytoplasmic domain of M2 at positions 71 and 78 or at the N terminus of the M1 protein at residues 31 and 41. To further examine the genetic basis for sensitivity and resistance to the 14C2 antibody, the nucleotide sequences of RNA segment 7 of several natural isolates of influenza virus have been obtained. Differences in the M1 and M2 amino acid sequences for some of the naturally resistant strains correlate with those found for the M2 antibody variant viruses. The possible interaction of M1 and M2 in virion assembly is discussed.

Nucleotide sequence of RNA segment 7 and the predicted amino sequence of M1 and M2 proteins of FPV/Weybridge (H7N7) and WSN (H1N1) influenza viruses

Since the gene products (M1 and M2) of influenza virus RNA segment 7 have been implicated in host range restriction, sensitivity to the drug amantadine, virus yield in chicken embryos as well as in virus assembly and morphology, we have determined the nucleotide sequence of this RNA segment for an avian [A/FPV/Weybridge (H7N7)] and a human [A/WSN/33 (H1N1)] virus and compared it to that of other influenza A virus strains. The results show that all ten strains of influenza A virus contain an identical number of nucleotides (1027 bases) in RNA segment 7 and an identical number of amino acids in Ml (252 aa) and M2 (97 aa) proteins. The observed amino acid changes are conservative in nature suggesting the requirement of a critical structure of both proteins in virus assembly. Furthermore, the presence of some consistent amino acid substitutions among different human and avian virus strains also supports the possible existence of host range and drug resistance determinants in M1 and M2 proteins.

Nucleotide sequence of RNA segment 7 of influenza B/Singapore/222/79: Maintenance of a second large open reading frame

The nucleotide sequence of RNA segment 7 of influenza B/Singapore/222/79 has been determined. The deduced amino acid sequence of the M 1 protein indicates that it contains 248 amino acids of which 243 are identical in the B/Lee/40 M, protein. A second overlapping open reading frame with a maximum coding region of 195 amino acids has also been maintained in the 39 years separating the two isolates. The deduced amino acid sequence of the second coding region displays 86% homology with the comparable B/Lee/40 sequence (27 changes). The conservation of the second open reading frame suggests that this region is important in the replicative cycle of influenza B viruses.

Nucleotide sequence analysis of the nucleoprotein gene of an avian and a human influenza virus strain identifies two classes of nucleoproteins

The nucleotide sequences of RNA segment 5 of an avian influenza A virus, A/Mallard/ NY?6750/78 (H2N2), and a human influenza A virus, A/Udorn/307/72 (H3N2), were determined and the deduced amino acid sequences of the nucleoprotein (NP) of these viruses were compared to two other avian and two other human influenza A NP sequences. The results indicated that there are separate classes of avian and human influenza A NP genes that can be distinguished on the basis of sites containing amino acids specific for avian and human influenza viruses and also by amino acid composition. The human influenza A virus NP genes appear to follow a linear pathway of evolution with the greatest homology (96.9%) between A/NT/60/68 (H3N2) and A/Udorn/72, isolated only 4 years apart, and the least homology (91.1 %)between A/PR/8/34 (H1N1) and A/Udorn/72, isolated 38 years apart. Furthermore, 84% of the nucleotide substitutions between A/PR/8/34 and A/NT/ 60 68 are preserved in the NP gene of the A/Udorn/72 strain. In contrast, a distinct linear pathway is not present in the avian influenza NP genes since the homology (90.3%)between the two avian influenza viruses A/Parrot/Ulster/73 (H7N1) and A/Mallard/78 isolated only 5 years apart is not significantly greater than the homology (90.1 %)between strains A/FPV/Rostock/34 and A/Mallard/78 isolated 44 years apart and only 49% of the nucleotide substitutions between A/FPV/34 and A/Parrot/73 are found in A/Mallard/78. A determination of the rate of evolution of the human influenza A virus NP genes suggested that there were a greater number of nucleotide substitutions per year during the first several years immediately following the emergence of anew subtype in 1968.

Influenza C virus RNA 7 codes for a nonstructural protein.

The complete nucleotide sequence of RNA segment 7 of influenza C/California/78 virus was determined by using cloned cDNA derived from viral RNA. The gene is 934 nucleotides long and possesses a long open reading frame which can code for a protein of 286 amino acids. Hybrid arrest translation experiments with the cloned cDNA fragment and poly(A)-containing RNA isolated from virus-infected cells showed that a 28,500-molecular-weight protein is coded for by RNA 7. Comparison of the proteins induced in the cell-free system and in virus-infected cells with those found in purified virus suggests that the 28,500-molecular-weight protein is a nonstructural protein.

Nucleotide sequence of RNA segment 5, encoding the nucleoprotein, of influenza A/FPV/Rostock/34

The nucleotide sequence of a cloned DNA copy of genome RNA segment 5 of fowl plague virus (influenza A/FPV/Rostock/34) has been determined. Segment 5 is 1565 nucleotides in length and codes for a nucleoprotein of 498 amino acids. Comparison of the sequence with the nucleoproteins of two human influenza A strains reveals 84–85% homology with the avian strain at the nucleotide level but 91–93% homology at the amino acid level. This presumably reflects common functional requirements of influenza A nucleoproteins.

Complete nucleotide sequence of the influenza C/California/78 virus nucleoprotein gene

The complete nucleotide sequence of RNA segment 5 of the influenza C/California/78 (C/Cal/78) virus was determined by using cloned cDNA derived from viral RNA. The gene contains 1809 nucleotides and can code for a protein of 565 amino acids with a molecular weight of 63 525. The RNA 5 protein of the influenza C/Cal/78 virus possesses two short regions which share a high degree (60–83%) of sequence homology with the nucleoproteins of influenza A and B viruses. These and other structural features of the RNA 5 protein suggest that RNA 5 of influenza C viruses codes for the nucleoprotein. The data also suggest that influenza C viruses are orthomyxoviruses, but that they are more distantly related to either type A or type B viruses than are influenza A and B viruses to each other.

Nucleotide sequence of RNA segment 3 of the avian influenza A/FPV/Rostock/34 and its comparison with the corresponding segment of human strains A/PR/8/34 and A/NT/60/68

The nucleotide sequence of RNA segment 3 of A/FPV/Rostock/34 (H7N1), an avian strain of influenza A virus, has been determined from a cloned DNA copy. Segment 3 codes for the PA polypeptide and the sequence specifies an acidic polypeptide of 716 amino acid residues. Comparison of the sequence with the corresponding segment of two human strains A/PR/8/34 and A/NT/60/68 indicates significant divergence of the avian sequence from the human sequences at the nucleotide level. At the amino acid level there is considerably greater homology between the avian and human strains. This presumably reflects a constraint on divergence of the PA polypeptide imposed by a common functional requirement of PA in all influenza virus strains.

Structure of the influenza C glycoprotein gene as determined from cloned DNA

The complete nucleotide sequence of RNA segment 4 of influenza C/JHB/1/66 virus has been determined, utilizing cloned cDNA derived from the viral RNA segment. The gene is 2073 nucleotides in length, and can code for a polypeptide of 655 amino acids, which corresponds to the viral glycoprotein. The predicted polypeptide has a molecular weight of 72063, not including oligosaccharides linked to eight predicted glycosylation sites. The influenza C glycoprotein shares structural features with hemagglutinin (HA) glycoproteins of influenza A and B viruses, including three stretches of hydrophobic amino acids believed to function as a signal sequence, a fusion function, and a membrane anchor. However, a substantial part of the protein lacks direct sequence homology to these HA glycoproteins. The results suggest a more distant evolutionary relationship between influenza C virus and influenza A and B viruses, compared to that between influenza A and B themselves.

Nucleotide sequence of human influenza A/PR/8/34 segment 2.

The nucleotide sequence of RNA segment 2 of human influenza strain A/PR/8/34 has been determined. Segment 2 in 2341 nucleotides long and encodes a protein of 757 amino acids (86,500 daltons molecular weight) which is involved in RNA synthesis. Although segment 2 is identical in size to segment 1, which encodes a protein of related function, neither the nucleotide sequences of these two RNA segments nor the amino acid sequences of the encoded proteins appear to be homologous. The sequence of segment 2 completes the sequence of the virus (total 13,588 nucleotides).

Identification of a second protein (M 2) encoded by RNA segment 7 of influenza virus

Recent analyses of the nucleotide sequences of RNA segment 7 of influenza virus from two strains of infuenza A virus (PR/8/34 and Udorn/72) have shown that in addition to the reading frame coding for the membrane protein (M 1) there is a second overlapping open reading frame that could code for 97 amino acids [ Winter and Fields (1980). Nucleic Acids Res. 8, 1965–1974; Allen et al. (1980). Virology 107, 548–551; Lamb and Lai (1981). Virology 112, 746–751 ]. We have identified such a protein (M 2) in infected cells and shown that it is synthesized from a separate mRNA. Hybrid-arrested translation experiments and the use of recombinant viruses of defined genome composition have demonstrated by both biochemical and genetic approaches that protein M 2 is coded for by RNA segment 7. Peptide mapping studies have shown that M 1 and M 2 are distinct proteins, and the peptide maps can be correlated with the nucleotide sequences coding for M 1 and M 2.

Influenza virus RNA segment 7 has the coding capacity for two polypeptides

The genes of a recombinant human influenza virus A/Japan/305/57:A/Bel/42:A/PR/8/34 have been cloned in bacterial plasmids, and the nucleotide sequence of RNA segment 7 has been determined. The DNA sequence predict two open reading frames, which overlap by 68 bases and have a coding capacity for polypeptides of 27,000 and 11,000 daltons. Segment 7 has been previously assigned as the gene coding for the viral matrix protein of apparent molecular weight 25,000. Therefore gene 7 has the potential to code for a previously unsuspected protein of approximately 11,000 daltons in addition to matrix.