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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 10539-10543, November 1993 Microbiology RNA sequence of astrovirus: Distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis BAOMING JIANG*t, STEPHAN S. MONROE*, EUGENE V. KOONINt, SARAH E. STINE*, AND ROGER I. GLASS* *Viral Gastroenteritis Section, Centers for Disease Control and Prevention, Atlanta, GA 30333; and tNational Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20894 Communicated by Bernard Fields, July 27, 1993 ABSTRACT The genomic RNA of human astrovirus was In the present study, we have sequenced and analyzed the sequenced and found to contain 6797 nt organized into three entire genomic RNA of H-Ast2§ and compared the sequence open reading frames (la, lb, and 2). A potential ribosomal and genomic structure with those of other positive-strand frameshift site identified in the overlap region of open reading RNA viruses. The results highlight the specific genomic frames la and lb consists ofa "shifty" heptanucleotide and an organization of astroviruses and support their classification RNA stem-loop structure that closely resemble those at the in a separate virus family. gag-pro junction of some retroviruses. This translation frame- shift may result in the suppression of in-frame amber termi- nation at the end of open reading frame la and the synthesis of MATERIALS AND METHODS a nonstructural, fusion polyprotein that contains the putative Cells and Virus. LLCMK2 cells (ATCC CCL 7.1) were protease and RNA-dependent RNA polymerase. Comparative propagated in Earle's minimal essential medium (MEM) sequence analysis indicated that the protease and polymerase of supplemented with antibiotics-and 10% fetal bovine serum. astrovirus are only distantly related to the respective enzymes H-Ast2 was obtained from John Kurtz (Oxford, England) and of other positive-strand RNA viruses. The astrovirus polypro- used to infect LLCMK2 cells in MEM/trypsin at 5 pg/ml as tein lacks the RNA helicase domain typical of other positive- described (2). Virions were partially purified from infected strand RNA viruses of similar genome size. The genomic cell lysates by centrifuging through a 30% (wt/vol) sucrose organization and expression strategy of astrovirus, with the cushion, suspension in TNE buffer [0.05 M Tris (pH 7.5)/0.1 protease and the polymerase brought together by predicted M NaCl/5 mM EDTA]/1% SDS, and extraction with phenol/ frameshift, most dosely resembled those of plant luteoviruses. chloroform. Virion RNA was precipitated with 2 M LiCl and Specific features of the sequence and genomic organization used for both sequencing and PCR assays. support the classification ofastroviruses as an additional family cDNA Synthesis and Sequencing. Single-stranded cDNA of positive-strand RNA viruses, designated Astroviridae. was synthesized from virion RNA with Super reverse tran- scriptase (Molecular Genetics Resources, Tampa, FL) by Astroviruses were originally identified from the feces of using primers derived originally from cDNA sequence (8) and infants with gastroenteritis on the basis of distinctive ultra- subsequently from sequences determined by directly se- structural features: five- or six-pointed surface stars are quencing virion RNA, using a "primer walking" technique. characteristic of this agent (1). These nonenveloped agents DNA fragments ofvarious length were amplified by the PCR were subsequently determined to be positive-strand RNA assay with Taq polymerase (Perkin-Elmer) and virus-specific viruses (2, 3). Five serotypes have been defined to date based primers. Sequences were determined from three sources: on their distinct antigenicity (4). Astroviruses cause acute virion RNA, PCR DNA, and cDNA clones (8). Virion RNA gastroenteritis in children and adults worldwide (5), but the was directly sequenced by an RNA disease burden has been difficult to determine because of the using sequencing kit lack of sensitive diagnostic assays. Recent studies have (Boehringer Mannheim). Both the PCR DNA and the cloned shown that astroviruses are more frequently found in children cDNA were sequenced by using the Sequenase version 2.0 with diarrhea than was previously thought (6). In addition, DNA sequencing kit (United States Biochemical). Sequences astroviruses have been detected in the diarrheal feces of on both strands of DNA were determined with each base various animals (5). sequenced at leastfourtimes. Sequences were assembled and Studies of the biochemical properties of purified particles aligned by using the Genetics Computer Group sequence- have provided divergent results' on the number and size of analysis package (9), and a consensus sequence was derived. proteins in astroviruses; two to six polypeptides have been Sequences of the 5' and 3' ends of the genomic RNA were reported, ranging in size from 5.5 kDa to 42 kDa (5). Although determined by following the procedure of Lambden et al. the fastidious growth of astroviruses in vitro has hindered (10). Briefly, a synthetic primer 1 was ligated to the 3' ends characterization of the genome, several investigators (3, 7) ofvirion RNA or cDNA corresponding to the 5' end ofvirion have reported partial sequence information from both inter- RNA with T4 RNA ligase (GIBCO/BRL). cDNA fragments nal regions and the 3' end of human astrovirus serotype 1 (400-600 bp) spanning either the 5' or the 3' ends were (H-Astl), and we have recently sequenced and characterized produced by PCR amplification using a primer 2 complemen- the subgenomic RNA of serotype 2 (H-Ast2; ref. 8). How- ever, the complete sequence and the genomic organization of Abbreviations: H-Ast2, human astrovirus serotype 2; RdRp, RNA- astroviruses remained unknown, and their classification was dependent RNA polymerase; NLS, nuclear localization signal; ORF, tentative. open reading frame; RHDV, rabbit hemorrhagic disease virus. tTo whom reprint requests should be addressed at: Mailstop G04, Centers for Disease Control and Prevention, 1600 Clifton Road, The publication costs of this article were defrayed in part by page charge Atlanta, GA 30333. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L13745). 10539 Downloaded by guest on October 1, 2021 10540 Microbiology: Jiang et al. Proc. Natl. Acad. Sci. USA 90 (1993) 5' 3, Genome I AA 1 6797 Subgenomic RNA AA 4314 6797 ORF lb 2773 Pol 4329 ORF 2 ORF la V RFS _ _ -- 1 L - i~~~~~~~~~~~~~~~~~~~~~~ NLS MB Pro 4325 6712 3 MB 2842 . ... .... I I. ....a I II ..I. I II I I IIIII I I I I. .....I III I a I a II I II . .. I ........ 0 1,000 2,000 3,000 4,000 5,000 6,000 Nucleotides FIG. 1. Genomic organization of human astrovirus. The locations of three ORFs, predicted transmembrane helices (MB), protease (Pro), nuclear localization signal (NLS), ribosomal frameshift structure (RFS), and RNA-dependent RNA polymerase (Pol) are indicated. ORFs la and lb encode a putative nonstructural polyprotein, and ORF 2 codes for a capsid-protein precursor. tary to the primer 1 and virus-specific primers and were groups of positive-strand RNA viruses-namely, animal sequenced by using internal primers. coronaviruses and arteriviruses, and plant luteoviruses and Comparative Sequence Analysis. Both nucleotide and de- dianthoviruses (19-22). However, the putative frameshifting duced amino acid sequences were compared by using the signal of astrovirus was much less similar to the frameshift BLAST program (11) and the BLOsUM62 matrix (12). Multiple regions of these viruses than to those of some retroviruses alignments were done by using the OPrAL or MACAW pro- (data not shown). The ribosomal frameshifting during trans- grams (13, 14). A phylogenetic tree was constructed by using lation ofastrovirus RNA probably directs the synthesis ofan clustering unweighted pairwise group maximum averages ORF la/lb fusion nonstructural polyprotein of 1416 aa with (UPGMA), neighbor-joining, least-square (Fitch-Margo- a predicted molecular mass of 161 kDa. liash), and protein-parsimony algorithms as implemented in The nucleotide sequence of the astrovirus genomic RNA the PHYLIP package (15). and the deduced amino acid sequences of the nonstructural polyprotein and the capsid protein of H-Ast2 were compared RESULTS AND DISCUSSION with partial sequences available for H-Astl. Between sero- The genomic RNA of H-Ast2 is 6797 nt in length, excluding types, the ribosomal frameshifting region was completely 31 adenines [poly(A) tail] at the 3' end. The genome possesses conserved (data not shown), and the amino acid sequence three overlapping open reading frames (ORFs la, lb, and 2; was highly conserved (>90%o identical) in a portion of the Fig. 1). The sequences surrounding the first AUG codons of nonstructural polyproteinsl (3) but was less conserved (51- ORFs la and 2 are predicted to be optimal for initiating 56% identical) in the predicted capsid-protein regions (3, 7). translation (16). ORF la is preceded by 82 untranslated Of interest, a region in the C-terminal portion of the non- nucleotides and encodes a polypeptide of 920 aa. Interest- structural polyprotein was significantly similar to the putative ingly, ORF lb, which overlaps ORF la by 70 nt, is in reading RNA-dependent RNA polymerases (RdRps) of plant bymo- frame +1, and its first AUG codon, which is predicted to be viruses (P = 0.015) and potyviruses (P = 0.095). This region weak, is located 380 nt downstream of the ORF la termina- contained the eight conserved motifs typical of the positive- tion codon. ORF 2, present also in the subgenomic RNA, strand RNA virus RdRps, indicating that it belongs to the overlaps ORF lb by 5 nt, begins with an initiation codon at so-called supergroup I, which includes the polymerases of nt 4325, and ends with a stop codon 82 bases from the 3' end.

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