1999

1999. p66, p46 and p120 were precursors of p28-p35 (NTPase), p32-p14 (VPg), and p32-p14 (VPg)-p70 (Pro-Pol), respectively. Mutagenesis in the Hoechst 34580 3C-like protease motif fully abolished the proteolytic activity. The cleavage map of SaV ORF1 is similar to those of additional heretofore known members of the family consists of four genera, (NoV; formerly known as Norwalk-like disease), and (SaV, formerly known as Sapporo-like disease), in which rabbit hemorrhagic disease disease (RHDV), feline calicivirus (FCV), Norwalk disease, and Sapporo disease are assigned as the prototype strains (8, 19). SaV is definitely associated with gastroenteritis in humans and swine (5, 9). Human being SaV is definitely mainly isolated from babies and young children, though it is occasionally associated with outbreaks of gastroenteritis (10, 21, 22, 33). Phylogenetic analysis using SaV capsid protein VP1 exposed five genetic organizations, genogroup I (GI) to GV (7). The human being SaV are classified into GI, GII, GIV, and GV, whereas porcine SaV belongs to GIII (7, 24, 26). Although porcine SaV can grow in cultured cells (4, 9), neither cell tradition nor animal models can support the replication of human being SaV. The SaV genome is definitely a positive-sense, single-strand RNA molecule of approximately 7.5 kb that is polyadenylated at its 3 terminus. The SaV GI, GIV, and GV genomes are expected to consist of three main open reading frames (ORFs), whereas SaV GII and GIII have two ORFs (9, 16, 22, 23, 25). The SaV ORF1 encodes nonstructural proteins and the capsid protein VP1, Hoechst 34580 while ORF2 and ORF3 encode proteins of yet unfamiliar function. To day, seven total genome sequences (accession figures in parentheses) of SaV, GI Manchester (“type”:”entrez-nucleotide”,”attrs”:”text”:”X86560″,”term_id”:”2437829″,”term_text”:”X86560″X86560), GI Dresden (“type”:”entrez-nucleotide”,”attrs”:”text”:”AY694184″,”term_id”:”51895344″,”term_text”:”AY694184″AY694184), GII Bristol (“type”:”entrez-nucleotide”,”attrs”:”text”:”AJ249939″,”term_id”:”18073224″,”term_text”:”AJ249939″AJ249939), and GII Mc10 (“type”:”entrez-nucleotide”,”attrs”:”text”:”AY237420″,”term_id”:”45545440″,”term_text”:”AY237420″AY237420), GII Sakai C12 (“type”:”entrez-nucleotide”,”attrs”:”text”:”AY603425″,”term_id”:”51243518″,”term_text”:”AY603425″AY603425), GIII PEC/Cowden (“type”:”entrez-nucleotide”,”attrs”:”text”:”AF182760″,”term_id”:”6164839″,”term_text”:”AF182760″AF182760), and PEC/LL14 (“type”:”entrez-nucleotide”,”attrs”:”text”:”AY425671″,”term_id”:”40037082″,”term_text”:”AY425671″AY425671) have been published. The SaV ORF1 polyprotein consists of amino acid motifs Hoechst 34580 characteristic of caliciviruses (8, 36), including 2C-like NTPase (NTPase) (GXXGXGKS/T), VPg [KGK(N/T)K and (D/E)EY(D/E)E], 3C-like protease (Pro)(GDCG), 3D-like RNA-dependent RNA polymerase (Pol)(GLPSG and YGDD), and VP1 (PPG) (9, 28). The proteolytic processing of ORF1 from the virus-encoded protease has been reported in RHDV, FCV, and NoV, and detailed cleavage maps of ORF1 Hoechst 34580 have been reported in these viruses (1, 20, 31, 36). A recent study having a full-length RNA transcript derived from the IGFIR SaV GI Manchester strain indicated six major cleavage products in vitro (6), suggesting that SaV ORF1 is also cleaved into nonstructural and structural proteins from the virus-encoded protease. However, neither the cleavage map of the viral proteins nor the function of the virus-encoded protease involved in this cleavage has been elucidated yet. In this study, the proteolytic control of the ORF1 polyprotein of SaV Mc10, a novel human being SaV GII strain, was analyzed by using an in vitro coupled transcription-translation system. We also evaluate the function of the virus-coded 3C-like protease on this proteolytic control. MATERIALS AND METHODS Specimen. The Mc10 strain (Hu/SaV/Mc10/2000/Thailand) was isolated in an epidemiological screening of acute gastroenteritis individuals in Thailand in June 2000 (10). The RNA extraction, reverse transcription-PCR, and total nucleotide sequence analysis of the disease genome were performed as previously explained (12). Building of plasmids comprising the full-length genome. A plasmid harboring the entire Mc10 genome was constructed with two overlapping cDNA fragments utilizing a unique HindIII site at nucleotides (nt) 3599 to 3604. The 5 fragment related to nt 1 to 3735 was amplified with a sense primer (5-GCGGGATCCTAATACGACTCACTATAGGgtgattggttagtatggcttccaagccattctacccaatag-3) including a BamHI site (underlined) and a T7 RNA polymerase promoter sequence (daring) and an antisense primer (5-TTGGGCCATGCAGGTGAGCG-3). The.