Analysis of M-class gene sequences and structural protein μA functions of avian reovirus

• Main Authors: Yu-Pin Su, 蘇育平
• Other Authors: 李龍湖
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/00682233641905785307

博士 === 中興大學 === 獸醫學系暨研究所 === 95 === The sequences and phylogenetic analyses of the M-class genome segments of 12 avian reovirus strains are described. The M1, M2 or M3 genome segment of S1133 strains is 2283, 2158 or 1996 base pairs long, respectively, encoding a protein μA, μB or μNS consisted of 732, 676 and 635 amino acids, respectively. The M1 genome segment has the 5′ GCUUUU terminal motif, but each M2 and M3 genome segment displays the 5′ GCUUUUU terminal motif which is common to other known avian reovirus genome segments. The UCAUC 3′-terminal sequences of the M-class genome segments are shared by both avian and mammalian reoviruses. Analysis of the average degree of the M-class gene and the deduced μ-class protein sequence identities indicated that the M2 genes and the μB proteins have the greatest level of sequence divergence. Computer searches revealed that the μA possesses a sequence motif (NH2-Leu-Ala-Leu-Asp-Pro-Pro-Phe-COOH) (residues 458-464) indicative of N-6 adenine-specific DNA methylase. Examination of the μB amino acid sequences indicated that the cleavage site of μB into μBN and μBC is between positions 42 and 43 near the N-terminus of the protein, and this site is conserved for each protein. During in vitro treatment of virions with trypsin to yield infectious subviral particles, both the N-terminal fragment δ and the C-terminal fragment φ were shown to be generated. The site of trypsin cleavage was identified in the deduced amino acid sequence of μB by determining the amino-terminal sequences of φ proteins: between arginine 582 and glycine 583. The predicted length of δ generated from μBC is very similar to that of δ generated from mammalian reovirus μ1C. Taken together, protein μB is structurally, and probably functionally, similar to its mammalian homolog, μ1. Phylogenetic analysis of the M-class genes revealed that the predicted phylograms delineated 3 M1, 5 M2, and 2 M3 lineages, no correlation with serotype or pathotype of the viruses. The results also showed that M2 lineages I–V consist of a mixture of viruses from the M1 and M3 genes of lineages I–III, reflecting frequent reassortment of these genes among virus strains. Analysis of amino acid sequence of core protein μA of avian reovirus has indicated that μA may share similar functions to protein μ2 of mammalian reovirus. Since μ2 displayed both NTPase and RTPase activities, μA was thus expressed in bacteria with a 4.5-kDa fusion peptide and six-His tag at its N-terminus. The purified recombinant μA (rμA) was used to test its NTPase and RTPase activities. Results indicated that rμA possessed NTPase activity that enabled the protein to hydrolyze the β-γ phosphoanhydride bond of all four NTPs since NDP was the only radiolabeled product. The substrate preference was ATP > CTP > GTP > UTP, based on the estimated kcat values. Alanine substitutions for lysines 408 and 412 (K408A/K412A) in a putative nucleotide binding site of rμA abolished NTPase activity, further suggesting that NTPase activity is attributable to protein rμA. The activity of rμA is dependent on the divalent cations Mg2+ or Mn2+, but not Ca2+ or Zn2+. Optimal pH for NTPase activity of rμA was achieved between pH 5.5 and 6.0. In addition, rμA enzymatic activity increased with temperature until 40 °C and was almost totally inhibited at temperature higher than 55 °C. Tests of phosphate release from RNA substrates with rμA or K408A/K412A rμA indicated that rμA but not K408A/K412A rμA displayed RTPase activity. The results suggested that both NTPase and RTPase activities of rμA might be carried out at the same active site and protein μA would play important roles during viral RNA synthesis.