Impact of the variations in potential glycosylation sites of the hemagglutinin of H9N2 influenza virus

Variations in the potential glycosylation sites were observed in hemagglutinin (HA) sequences of H9N2 avian influenza virus isolated in China, deposited in the Influenza Virus Resource of NCBI before 2017, which showed a deleted glycosylation site at amino acid residue 218, and an introduced glycosylation site at amino acid residue 313. Based on the variations in the glycosylation sites at these amino acids, H9N2 avian influenza viruses could be divided into three categories. Firstly, most of the H9N2 influenza viruses were 218G⁺ viruses; less 313G⁺ viruses were isolated between 1997 and 2004. Secondly, the occurrence of the 218G⁺/313G⁺ viruses increased, while the 218G⁺/313G^? viruses decreased from 2005 to 2012. Thirdly, from 2013 to 2016, the 218G^?/313G⁺ viruses were predominant compared to the 218G⁺/313G⁺ viruses. Here, based on an F/98 virus backbone, a 218G⁺/313G^? virus, two reassortment viruses were generated, and named rF/HA218G⁺/313G⁺ and rF/HA 218G⁺/313G^?, respectively. HA protein migration demonstrated that the glycosylation sites at amino acid residues 313 and 218 were both functional. The absence of the glycosylation site at amino acid residue 218 and the presence of the glycosylation site at amino acid residue 313 increased antibody binding and moderately prevented the virus from escaping neutralization with homologous antisera. Additionally, compared to the F/98 virus (218G⁺/313G^?), the viruses rF/HA218G⁺/313G⁺ or rF/HA218G^?/313G⁺ showed significantly increased infectivity of MDCK cells, chicken embryo eggs, and trachea and lung tissue of SPF chickens, but did not display differences in airborne spread in chickens or infectivity of mice compared with its parental virus F/98.

     

Coding properties of the S and the M genome segments of Sapporo rat virus: comparison to other causative agents of hemorrhagic fever with renal syndrome

Three serologically distinct groups of hantaviruses have been associated with severe, moderate, and mild forms of hemorrhagic fever with renal syndrome (HFRS). To gain a better understanding of the genetic variation among these viruses, we cloned and sequenced the M and the S genome segments of Sapporo rat virus, an etiologic agent of moderate HFRS, and compared the predicted gene products to those of Hantaan virus, and the H?lln?s strain of Puumala virus, which are etiologic agents of severe and mild HFRS, respectively. The SR-11 S segment consisted of 1769 nucleotides and had an open reading frame (ORF) in the virus-complementary sense RNA with a coding capacity of 429 amino acids. Deduced amino acids from the SR-11 S segment ORF displayed 83% homology with those of Hantaan nucleocapsid (N) protein. Comparison of the S segment ORFs of all three viruses revealed 58% homology. No evidence for additional nonstructural protein(s) encoded by the SR-11 S segment was obtained. The SR-11 M segment consisted of 3651 nucleotides and had an ORF in the virus-complementary sense RNA with a coding capacity of 1134 amino acids. Amino acid sequences predicted from the SR-11 M segment ORF were 75% homologous with those encoding Hantaan G1 and G2 envelope glycoproteins. Comparison of the deduced amino acid sequences of the M segment ORFs of SR-11, Hantaan, and H?lln?s viruses revealed a 43% homology for amino acids constituting the G1 proteins and a 55% homology for amino acids constituting the G2 proteins of the three viruses. The envelope proteins of SR-11 virus were localized within the M segment ORF by amino-terminal sequence analysis of purified G1 and G2. G1 initiated at amino acid 17 and G2 at amino acid 647 within the ORF. Five potential asparagine-linked glycosylation sites were identified in the SR-11 G1 coding sequences, four of which were conserved between Hantaan and SR-11 viruses and three of which were conserved among all three viruses. One potential glycosylation site was identified in the SR-11 G2 coding sequences and was conserved among Hantaan, SR-11 and H?lln?s viruses. Cysteine residues were highly conserved within the M segment ORFs of all three viruses, suggesting a similar structure and function of the G1 and G2 proteins.     

Genetic analysis of an influenza B virus isolated from a patient with encephalopathy in Japan

An influenza B virus, B/Saga/S172/99 (SAG99), was isolated from the nasopharynx of a patient with encephalopathy/encephalitis in Japan in 1999. To clarify the molecular characteristics of this virus, detailed analysis of the gene segments coding for the hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (M) and non-structural protein (NS) was undertaken. All five genes of SAG99 showed high nucleotide and predicted amino acid similarities with those of recent non-encephalopathic strains isolated in the same epidemic season. Subsequent phylogenetic analysis revealed that all five gene segments of SAG99 analyzed in the present study were most similar to those of the recent Yamagata/16/88-like viruses. The hemagglutinin and neuraminidase proteins of SAG99 were each distinguished from those of recent epidemic strains by one characteristic amino acid substitution. These substitutions were not found in the previously reported encephalopathy/encephalitis-derived influenza B viruses, and we could not find any common characteristic amino acid changes in SAG99 and these viruses. Similarly, among the internal proteins studied, only the M2 protein of SAG99 was found to contain a single novel amino acid change when compared with other recent isolates. Thus, it was apparent that SAG99 contained very few amino acid differences when compared with other epidemic viruses. The association of recent B/Yamagata/16/88-like viruses with encephalitis/encephalopathy observed in the present study and previously suggest that these viruses may have a higher potential for causing neurological complications in certain individuals.     

一株鹅H5N1亚型流感病毒基因特性的分析

目的 弄清了Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）毒株对鹅致病的分子生物学基础 ，研究香港区人群中发生的禽（Ｈ５Ｎ１）流感的病因，方法 病毒ＲＮＡ经逆转录合成ｃＤＮＡ经聚合酶链反应（ＰＣＲ）扩增，产物纯化，采用双脱链末端终止法测定核苷酸序列，结果 Ａ／鹅／广东／２／９６（Ｈ５Ｎ１）与Ａ／ＨＫ／１５６／９７（Ｈ５Ｎ１）毒株ＲＮＡ４核苷酸序列有２２个位点不同（同源性为９８．８％）无任何掉失或插入。它与人和     

深圳地区甲1(H1N1)亚型流感病毒基因特性的研究

目的　了解近几年H1N1亚型毒株在深圳地区人群中活动加强及“O”相特性出现的分子生物学基础及其基因演变的特性。方法　病毒粒RNA经逆转录合成cDNA,用PCR扩增,产物纯化,采用双脱氧链末端终止法进行核苷酸序列测定并推导出其所编码的氨基酸序列。进化树分析用DNASTAR公司出品的序列分析软件,MegAlign(103版)的Editseq(369版)。结果　根据病毒粒HA1基因特性,至少1995年以来深圳地区人群中同时流行着基因特性不同的三系毒株;1995～1997年H1N1毒株与A新加坡686(H1N1)病毒相比较,其HA1蛋白分子上第54和155位上分别插入和缺失一个糖基化点,同时氨基酸序列发生了替换。结论　近年来深圳地区人群中同时流行着HA1基因不同的三系H1N1亚型毒株。由于HA1蛋白分子上氨基酸序列发生了替换,尤其糖基化位点插入和缺失,造成1995年以来H1N1毒株活动加强,这些可能与毒株“O”相特性再现密切相关。     

Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin?

The A/Chick/Penn/83 (H5N2) influenza virus that appeared in chickens in Pennsylvania in April 1983 and subsequently became virulent in October 1983, was examined for plaque-forming ability and cleavability of the hemagglutinin (HA) molecule. The avirulent virus produced plaques and cleaved the HA only in the presence of trypsin. In contrast, the virulent virus produced plaques and cleaved the HA precursor into HA1 and HA2 in the presence or absence of trypsin. The apparent molecular weight of the HA1 from the avirulent virus was higher than that from the virulent virus, but when the viruses were grown in the presence of tunicamycin, the molecular weights of HA were indistinguishable. Two of nine monoclonal antibodies to the HA of the avirulent virus indicate that there is at least one epitope on the HA that is different between the virulent and avirulent viruses. The amino acid sequences of the HAs from the two viruses were compared by sequencing their respective HA gene. The nucleotide sequence coding for the processed HA polypeptide contained 1641 nucleotides specifying a protein of 547 amino acids. The amino acid sequences of the virulent and avirulent viruses were indistinguishable through the connecting peptide region, indicating that the difference in cleavability of the H5 HA is not directly attributed to the amino acid sequence of the connecting peptide. Four of seven nucleotide changes resulted in amino acid changes at residues 13, 69, and 123 of HA1 and at residue 501 of the HA2 polypeptide. Since there were no deletions or insertions in the amino acid sequence of the virulent or avirulent viruses, the possibility exists that the difference in molecular weight is due to loss of a carbohydrate side chain in the virulent strain. The amino acid change in the virulent strain at residue 13 is the only mutation that could affect a glycosylation site and this is in the vicinity of the connecting peptide. It is postulated that the loss of this carbohydrate may permit access of an enzyme that recognizes the basic amino acid sequences and results in cleavage activation of the HA in the virulent virus.     

Migration of the Swine Influenza Virus δ-Cluster Hemagglutinin N-Linked Glycosylation Site from N142 to N144 Results in Loss of Antibody Cross-Reactivity

Routine antigenic characterization of swine influenza virus isolates in a high-throughput serum neutralization (HTSN) assay found that approximately 20% of isolates were not neutralized by a panel of reference antisera. Genetic analysis revealed that nearly all of the neutralization-resistant isolates possessed a seasonal human-lineage hemagglutinin (HA; δ cluster). Subsequent sequencing analysis of full-length HA identified a conserved N144 residue present only in neutralization-resistant strains. N144 lies in a predicted N-linked glycosylation consensus sequence, i.e., N-X-S/T (where X is any amino acid except for proline). Interestingly, neutralization-sensitive viruses all had predicted N-linked glycosylation sites at N137 or N142, with threonine (T) occupying position 144 of HA. Consistent with the HTSN assay, hemagglutination inhibition (HI) and serum neutralization (SN) assays demonstrated that migration of the potential N-linked glycosylation site from N137 or N142 to N144 resulted in a >8-fold decrease in titers. These results were further confirmed in a reverse genetics system where syngeneic viruses varying only by predicted N-glycosylation sites at either N142 or N144 exhibited distinct antigenic characteristics like those observed in field isolates. Molecular modeling of the hemagglutinin protein containing N142 or N144 in complex with a neutralizing antibody suggested that N144-induced potential glycosylation may sterically hinder access of antibodies to the hemagglutinin head domain, allowing viruses to escape neutralization. Since N-linked glycosylation at these sites has been implicated in genetic and antigenic evolution of human influenza A viruses, we conclude that the relocation of the hemagglutinin N-linked glycosylation site from N142 to N144 renders swine influenza virus δ-cluster viruses resistant to antibody-mediated neutralization.     

猪型流感病毒血凝素基因的核苷酸全序列分析

对我国大陆首次从猪群中分离到的猪型（Ｈ１Ｎ１）流感病毒血凝素（ＨＡ）基因核苷酸全序列进行了测定，其长度为１７７８ｂｐ，共编码５６６个氨基酸，其中信号区１７个，ＨＡ１区３２６个，多肽连接区１个，ＨＡ２区２２２个。与Ａ／ＮＪ／１１／７６（Ｈ１Ｎ１）毒株ＨＡ蛋白分子上氨基酸序列相比，其同源性多１个糖基化点，然而其余的包括２个重叠的糖基化点均相同。     

Nucleotide sequence analysis of the bovine respiratory syncytial virus fusion protein mRNA and expression from a recombinant vaccinia virus

Bovine respiratory syncytial (BRS) virus is an important cause of serious respiratory illness in calves. The disease caused in calves is similar to that caused by human respiratory syncytial (HRS) virus in children. The two viruses, however, have distinct host ranges and the attachment glycoproteins, G, have no antigenic cross-reactivity. The fusion glycoproteins, F, of the HRS and BRS viruses, however, have some antigenic cross-reactivity. To further compare the BRS virus and HRS virus fusion proteins, we determined the nucleotide sequence of cDNA clones to the BRS virus F protein mRNA, deduced the amino acid sequence, and compared these sequences with the HRS virus F protein sequences. The BRS virus F mRNA was 1899 nucleotides in length and had a single major open reading frame which could code for a polypeptide of 574 amino acids with an estimated molecular weight of 63.8 kDa. Structural features predicted from the amino acid sequence included an NH2-terminal signal sequence (residues 1-26), a site for proteolytic cleavage (residues 131-136) to generate the disulfide-linked F1 and F2 subunits, and a hydrophobic transmembrane anchor sequence (residues 522-549). The nucleic acid identity between the BRS virus and the HRS virus F mRNA sequences was 71.5%. The predicted BRS virus F protein shared 80.5% overall amino acid identity with the HRS virus F protein with 89% identity in the F1 polypeptide but only 68% identity in the F2 polypeptide. The position and number of the cysteine residues in the F1 and F2 polypeptides were conserved among all F proteins. However, BRS virus F protein had only three potential N-linked carbohydrate acceptor sites in comparison to four or five for the HRS viruses. A difference in the extent of glycosylation between the BRS and HRS virus F2 polypeptides was shown to be responsible for differences observed in the electrophoretic mobility of these proteins. A cDNA containing the complete open reading frame of the BRS virus F mRNA was inserted into the thymidine kinase gene of vaccinia virus and following homologous recombination, a recombinant virus containing the BRS virus F gene was isolated. The BRS virus F protein was expressed in recombinant virus infected cells as demonstrated by immunoprecipitation and was transported to and expressed on the surface of infected cells as shown by indirect immunofluorescence.     

Isolation and molecular characterization of equine H3N8 influenza viruses from pigs in China

During 2004–2006 swine influenza virus surveillance, two strains of H3N8 influenza viruses were isolated from pigs in central China. Sequence and phylogenetic analyses of eight gene segments revealed that the two swine isolates were of equine origin and most closely related to European equine H3N8 influenza viruses from the early 1990s. Comparison of hemagglutinin (HA) amino acid sequences showed several important substitutions. One substitution caused the loss of a potential glycosylation site, and two substitutions, located at the cleavage site and adjacent to the receptor-binding pocket, respectively, had been reported previously in canine H3 HAs. This expansion of host range of equine H3N8 influenza viruses with mutations in the HA protein might raise the possibility of transmission of these viruses to humans. J. Tu and H. Zhou contributed equally to the work.     

Pneumotropic revertants derived from a pantropic mutant, F1-R, of Sendai virus.

Revertants were isolated from the protease activation mutant of Sendai virus, F1-R, which causes a systemic infection in mice. The fusion (F) glycoprotein of F1-R is susceptible to activation cleavage by ubiquitous cellular proteases and is thus responsible for pantropism in mice (Tashiro et al., 1988. Virology 165, 577-583). The revertants regained several phenotypes of wild-type virus; they required exogenous trypsin for activation of the F protein in cell cultures and in nonpulmonary mouse tissues and they were exclusively pneumotropic in mice. On the other hand, phenotypes of F1-R that remained unchanged by the revertants were bipolar budding in polarized epithelial cells, enhanced electrophoretic migration of the matrix protein, and the lack of a glycosylation site in the F2 subunit of the F protein. Comparative RNA sequence analysis of the F gene of the revertants revealed that the reduced cleavability of the F protein of the revertants was the result of the predicted single amino acid reversion (Pro to Ser) at residue 115 adjacent to the cleavage site. Thus the sequence at the cleavage site of the revertants was Ser-Lys compared with Pro-Lys for F1-R and Ser-Arg for wild-type virus. The results indicate that enhanced cleavability of the glycoprotein, a feature often associated with multiple basic residues within the cleavage site of paramyxovirus F proteins and influenza virus hemagglutinins, can also be determined by a single basic amino acid following proline. Additionally, the revertants were less susceptible to the activator for wild-type virus present in mouse lungs and less pathogenic for this organ than wild-type virus. These results provide further evidence that proteolytic activation of the F protein by host proteases is the primary determinant for organ tropism and pathogenicity of Sendai virus in mice. One of the revertants was also temperature sensitive (ts); the ts lesion in the nucleoprotein gene was identical to that found in ts-f1, the ts host range mutant from which F1-R was derived.     

Evolutionary pattern of the H 3 haemagglutinin of equine influenza viruses: multiple evolutionary lineages and frozen replication

Summary The nucleotide and deduced amino acid sequences of the haemagglutinin genes coding for the HA 1 domain of H3N8 equine influenza viruses isolated over wide regions of the world were analyzed in detail to determine their evolutionary relationships. We have constructed a phylogenetic model tree by the neighbour-joining method using nucleotide sequences of 15 haemagglutinin genes, including those of five viruses determined in the present study. This gene tree revealed the existence of two major evolutionary pathways during a twenty five-year period between 1963 to 1988, and each pathway appeared to consist of two distinct lineages of haemagglutinin genes. Furthermore, our analysis of nucleotide sequences showed that two distinct lineages of equine H3N8 viruses were involved in an equine influenza outbreak during the period of December 1971–January 1972 in Japan. The number of nucleotide changes between strains was proportional to the length of time (in years) between their isolation except for three of the HA genes. However, there are three exceptional strains isolated in 1971, 1987, and 1988, respectively. The haemagglutinin gene in these strains showed a small number of nucleotide substitutions after they branched off around 1963, suggesting an example of frozen replication. Although the estimated rate (0.0094/site/year) of synonymous (silent) substitutions of the haemagglutinin gene of equine H3N8 viruses was nearly the same as that of human H 1 and H 3 haemagglutinin genes, the rate of nonsynonymous (amino-acid changing) substitutions of the former equine virus gene was estimated to be 0.00041/site/year — that is about 5 times lower than that estimated for the human H 3 haemagglutinin gene. The present study is the first demonstration that multiple evolutionary lineages of equine H3N8 influenza virus circulated since 1963.     

Impact of glycosylation on the immunogenicity of a DNA-based influenza H5 HA vaccine

Avian H5N1 influenza viruses isolated from humans in Hong Kong in 1997 were divided into two antigenic groups based on the presence or absence of a potential glycosylation site at amino acid residues 154-156 in the HA1 region of the viral hemagglutinin (HA) surface glycoprotein. To assess the impact of glycosylation on the immunogenicity of an HA-expressing DNA vaccine, a series of plasmid vaccine constructs that differed in the presence of potential glycosylation sites at amino acid residues 154-156, 165-167, and 286-288 were used to immunize BALB/c mice. Postvaccination serum IgG, hemagglutination inhibition, and neutralizing antibody titers as well as the morbidity and mortality following a lethal H5N1 viral challenge did not vary significantly among any of the experimental groups. We conclude that the glycosylation pattern of the influenza virus HA1 domain has little impact on the murine antibody response raised to a DNA vaccine encoding the H5 HA, thereby minimizing the concern that the pattern of glycosylation sites encoded by the vaccine match those of closely related H5 viruses.     

Genetic and antigenic variation in the haemagglutinin of recently circulating human influenza A (H3N2) viruses in the United Kingdom

Summary Variation in the haemagglutinin (HA) gene of influenza A (H₃N₂) viruses isolated in the U. K. and abroad from 1992–1994, was determined by nucleotide sequencing of the HA1 domain of the HA gene. Viruses isolated in the U.K. early in the 1992–93 season were from the A/Beijing/353/89 lineage and were replaced later that season by viruses from the A/Beijing/32/92 lineage. Viruses from the new lineage continued to be isolated during the 1993–94 season, but were heterogeneous. Most of these isolates were more closely related to an A/Beijing/32/92 variant, A Hong Kong/23/92, but could be distinguished into three groups by serology (of which one group was circulating during the previous season) and four groups based on sequence variation in the HA gene. However, phylogenetic analysis of antigenically-distinct isolates showed that the HA gene is evolving along one lineage. Sequence analysis identified mainstream, subgroup and strain specific amino acid substitutions. There was a broad correlation between the observed amino acid changes and the antigenic sites of the HA. The results of this study highlight the value of regular molecular analysis of circulating viruses.     

Genetic characterization of the hemagglutinin of two strains of influenza B virus co-circulated in Taiwan.

Two isolates of influenza B virus were obtained in the spring of 1997. One strain, B/Taiwan/21706/97, was isolated from a patient who had acute tonsillitis. The other, B/Taiwan/3143/97, was isolated from a patient who was diagnosed with meningoencephalitis. This implies that the influenza B viruses not only cause respiratory symptoms but may also cause inflammation of the nervous system. Sequence analysis of the hemagglutinin (HA) gene, HA1 domain, indicated that there were remarkable amino acid changes in the strain B/Taiwan/3143/97 compared to B/Victoria/2/87, B/Yamagata/16/88, and B/Taiwan/7/88. The changes in the positions 116, 200, 238, 242, and 271 were correlated with receptor binding. Furthermore, a potential glycosylation site at position 233 was lost. In total, 30 amino acid changes were noted at positions ranging from 116 to 295. These changes may affect the antigenicity of the virus. Phylogenetic analyses also showed that the B/Taiwan/3143/97 was located in an independent lineage, when compared to the reference strains belonging to B/Victoria/2/87 and B/Yamagata/16/88 lineages. This supports the hypothesis that influenza B viruses with distinct genetic characteristic were co-circulated in Taiwan.     

Molecular adaptation of an H7N3 wild duck influenza virus following experimental multiple passages in quail and turkey

To investigate the molecular adaptation of influenza viruses during natural interspecies transmission, we performed a phenotypic and genotypic analysis of a low-pathogenic duck H7N3 influenza virus after experimental passages in turkey and quail. Results obtained showed differences in the HA receptor-binding and in NA enzyme activities in viruses recovered after passages in quail, compared to those obtained from passages in turkey. Sequencing of the HA, NA and genes of internal proteins of the viruses obtained from quail and turkey, identified several amino acid substitutions in comparison with the progenitor virus. Of note, in the quail-adapted viruses the emergence of a 23-amino acid deletion in the stalk of the NA and the introduction of a glycosylation site in the HA were a reminiscence of changes typically observed in nature confirming a potential role of the quail in the adaptation of wild birds viruses to domestic poultry.     

甲1型流感病毒新分离株HA基因的序列分析

目的 研究新分离的H1N1亚型流感病毒株的HA1基因序列。方法 甲型流感病毒通过鸡胚增殖后提取RNA、逆转录合成cDNA,经PCR扩增和产物纯化构建重组质粒,用双脱氧链终止法进行核苷酸序列测定;并进行基因特性分析。结果 新分离到的3株流感病毒株（H1N1）HA1区基因长度为981bp,编码327个氨基酸;与A/桂防/10/94和A/Bayern/07/95（H1N1）标准株比较其同源性分别为92.8%和91.3%,丢失了第130位氨基酸和304位糖基化位点;新分离的3株甲型流感病毒（H1地）标准株比较其同源性分别为92.8%和91.3%,丢失了第130位氨基酸和304位糖基化位点,新分离的3株甲型流感病毒株（H1N1）HA1区氨基酸同源性高达98%;A/桂防/10/94和A/Bayern/07/95(H1N1)毒株HN1氨基酸的同源性高达96%。结论 新分离到的3株H1N1毒株HA编码氨基酸不同于A/Baydrn/07/95(H1N1)和A/桂防/10/94（H1N1）标准株,它们可能为新的甲型流感病毒变异性。     

Cocirculation of two distinct evolutionary lineages of influenza type B virus since 1983

During 1988-1989 two highly distinct antigenic variants of influenza type B were recognized in hemagglutination-inhibition tests with postinfection ferret serum. These viruses were antigenically related to either B/Victoria/2/87, the most recent reference strain, or B/Yamagata/16/88, a variant that was isolated in Japan in May 1988. All influenza B viruses isolated in the United States during an epidemic in the winter of 1988-1989 were antigenically related to B/Victoria/2/87. However, in several countries in Asia, both B/Victoria/2/87-like viruses and B/Yamagata/16/88-like viruses were isolated. Sequence analysis of the hemagglutinin (HA) genes of several influenza B isolates from 1987 to 1988 indicated that the HA1 domains of the B/Yamagata/16/88-like viruses and B/VI/87-like viruses isolated in 1988 differed by 27 amino acids. Evolutionary relationships based on this sequence data indicated that the B/Yamagata/16/88-like viruses were more closely related to epidemic viruses from 1983 (B/USSR/100/83-like viruses) than to more recent reference strains such as B/Victoria/2/87. All other Asian strains, as well as selected isolates from the United States in 1988, were confirmed by sequence analysis as being genetically related to B/Victoria/2/87. These data provide clear evidence that two parallel evolutionary pathways of influenza type B have existed since at least 1983 and that viruses from each of the separate lineages were isolated from cases of influenza B in 1988. This finding is similar to earlier observations for type A H1N1 and H3N2 influenza viruses.     

Cloning of the fusion gene of rinderpest virus: comparative sequence analysis with other morbilliviruses

We have cloned the cDNA of the fusion (F) gene of the virulent (Kabete O) strain of rinderpest virus and provided a comparative analysis of its sequence with that of the F genes of measles and distemper viruses. The gene has an open reading frame of 2241 nucleotides with two potential initiation codons in-frame. Use of the first ATG would produce a polypeptide 747 amino acids long with a calculated molecular weight of 81,068. However, we suggest that the second ATG is used to generate the Fo protein, which is 546 amino acids long with a calculated molecular weight of 58,754. During maturation, the cleavage of F0 gives rise to the functional F1 and F2 polypeptides. The F1 polypeptide is 438 amino acids long and has a calculated molecular weight of 46,791, with a single (potential) glycosylation site in its cytoplasmic domain. The F2 polypeptide, probably 89 amino acids long after the signal sequence is cleaved, is estimated to be 9,800 Da and has three potential glycosylation sites. There is a divergence of 18.7% in amino acid sequences between rinderpest and measles virus F0 polypeptides; between distemper and rinderpest viruses the divergence is 31.8%. No significant homology in nucleotide sequences of rinderpest DNA to measles or distemper DNA was found in the 5' and 3' untranslated regions.