Neutralization epitopes on rotavirus SA11 4fM outer capsid proteins.

The VP7 and VP4 genes of seven antigenic mutants of simian rotavirus SA11 4fM (serotype 3) selected after 39 passages in the presence of SA11 4fM hyperimmune antiserum, were sequenced. Nucleotide sequence analysis indicated the following. (i) Twice as many amino acid substitutions occurred in the VP7 protein than in VP4, which has a molecular weight twice that of VP7. (ii) Most amino acid changes that occurred clustered in six variable regions of VP7 and in two variable regions of VP4; these variable regions may represent immunodominant epitopes. (iii) Most amino acid substitutions that occurred in VP7 and VP4 of these mutants were also observed in antigenic mutants selected with neutralizing monoclonal antibodies (NMAbs); however, some amino acid substitutions occurred that were not selected for NMAbs. (iv) On VP7, some of the neutralization epitopes appeared to be interrelated because amino acid substitution in one site affected binding of specific NMAbs to other sites, while other neutralization epitopes on VP7 appeared to be independent, in that amino acid substitution in one site did not affect the binding of NMAbs to another distant site.     

Nucleotide sequence and corresponding amino acid sequence of the gene for the major antigen of foot and mouth disease virus

A segment of 1160 nucleotides of the FMDV genome has been sequenced using three overlapping fragments of cloned cDNA from FMDV strain O1K. This sequence contains the coding sequence for the viral capsid protein VP1 as shown by its homology to known and newly determined amino acid sequences from this man antigenic polypeptide of the FMDV virion. The structural gene for VP1 comprises 639 nucleotides which specify a sequence of 213 amino acids for the VP1 protein. The coding sequence is not flanked by start and stop codons which is consistent with the mode of biosynthesis of VP1 by post-translational processing of a polyprotein precursor.     

Molecular and antigenic characterization of porcine rotavirus YM, a possible new rotavirus serotype.

In 1983, we isolated a porcine rotavirus (strain YM) that was prevalent in several regions of Mexico, as judged by the frequency of its characteristic electropherotype. By a focus reduction neutralization test, rotavirus YM was clearly distinguished from prototype rotavirus strains belonging to serotypes 1 (Wa), 2 (S2), 3 (SA11), 4 (ST3), 5 (OSU), and 6 (NCDV). Minor, one-way cross-neutralization (1 to 5%) was observed when antisera to the various rotavirus strains were incubated with rotavirus YM. In addition, the YM virus was not neutralized by neutralizing monoclonal antibodies with specificity to serotypes 1, 2, 3, and 5. The subgroup of the virus was determined to be I by enzyme-linked immunosorbent assay. To characterize the serotype-specific glycoprotein of the virus at the molecular level, we cloned and sequenced the gene coding for VP7. Comparison of the deduced amino acid sequence with reported homologous sequences from human and animal rotavirus strains belonging to six different serotypes further supported the distinct immunological identity of the YM VP7 protein.     

Comparative analysis of the VP3 gene of divergent strains of the rotaviruses simian SA11 and bovine Nebraska calf diarrhea virus.

The gene encoding outer capsid protein VP3 of subpopulations of two animal rotaviruses, simian SA11 and Nebraska calf diarrhea virus (NCDV), was analyzed. Two laboratory strains of simian SA11 rotavirus (SA11-SEM and SA11-FEM) differed with respect to VP3. This dimorphism was indicated by a difference in electrophoretic mobility and a difference in reactivity with anti-VP3 monoclonal antibodies. The overall VP3 amino acid homology between the two SA11 VP3 proteins was 82.7%, whereas the VP3 protein of SA11-FEM was 98.5% homologous in amino acid sequence to NCDV VP3, suggesting that SA11-FEM VP3 was derived by gene reassortment in the laboratory during contamination with a bovine rotavirus. A comparison of the deduced amino acid sequence of the VP3 of two virulent NCDV strains and an attenuated NCDV strain (RIT 4237), revealed only five amino acid differences which were scattered throughout the protein but did not involve the trypsin cleavage sites. Of interest, the VP3 of the standard strain of NCDV which is virulent for cows differed in only one amino acid (position 23, Gln to Lys) from the VP3 of an NCDV mutant which was attenuated both for cows and for children.     

Rotavirus neutralizing protein VP7: antigenic determinants investigated by sequence analysis and peptide synthesis.

The rotavirus neutralizing antigen, VP7, is a 37,000-molecular-weight glycoprotein which is a major component of the outer shell of the virion. The amino acid sequence of VP7 for strain S2 (human serotype 2) and Nebraska calf diarrhea virus (bovine serotype) has been inferred from the nucleic acid sequence of cloned copies of genomic segment nine. Comparison of the amino acid sequences of these two VP7 proteins with those already determined for other rotavirus strains reveals extensive sequence conservation between serotypes with clusters of amino acid differences sited predominantly in hydrophilic domains of the protein. Six peptides have been synthesized that span the hydrophilic regions of the molecule. Antisera to these peptides both recognize the respective homologous peptides in a solid-phase radioimmunoassay and bind to denatured VP7 in a Western blot. However, none of the antisera either recognize virus or exhibit significant neutralizing activity, indicating that these peptide sequences are not available on the surface of the virus.     

Use of the polymerase chain reaction for analysis of rotaviruses. Nucleotide sequence of a gene, coding for the basic neutralizing antigen VR7 of a human rotavirus with a new G-serotype

A procedure based on polymerase chain reaction use for the detection of rotavirus has been developed. Full length cDNA copy of the VP7 gene coding for the major neutralization glycoprotein of the human rotavirus RK9 with an unusual "wide" electrophoretype is cloned and sequenced. Glycoprotein VP7 of RK9 has a unique amino acid composition in A and C antigenic regions. It shows that strain RK9 represents a new (12) rotavirus serotype.     

Cloning and nucleotide sequence of the simian rotavirus gene 6 that codes for the major inner capsid protein.

The nucleotide sequence of the gene that codes for the major inner capsid protein of the simian rotavirus SA11 has been determined. A DNA copy of mRNA from gene 6 was cloned in the E. coli plasmid pBR322. The full-length gene is 1357 nucleotides long with a 5'-noncoding region of 23 nucleotides and a 3'-noncoding region of 140 nucleotides. The gene contains a single, long, open reading-frame of 1194 nucleotides capable of coding for a protein of 397 amino acids with a molecular weight of 44,816. The predicted protein product is relatively proline-rich with a net charge at neutral pH of -3.5. One stretch of 53 amino acids (encoded by nucleotides 327-485) is basic.     

Complete nucleotide and derived amino acid sequences of the fourth component of mouse complement (C4). Evolutionary aspects

The nucleotide sequence coding for the fourth component of mouse complement (C4) has been determined from a cloned genomic DNA fragment and a cloned cDNA fragment. The amino acid sequence of the protein was deduced. The single chain precursor protein (pro-C4) consists of 1719 amino acid residues. The mature beta, alpha, and gamma subunits contain 654, 766, and 291 amino acids, respectively. One potential carbohydrate attachment site is predicted for the beta chain, three for the alpha chain, and none for the gamma chain. From a comparison with human C4 cDNA sequence an extensive overall sequence homology, 79% in nucleotides and 76% in amino acids, is observed. There is conservation in both the position and number of cysteine residues in human and mouse C4. We compared the mouse C4 amino acid sequences with those of mouse C3 and human alpha 2-macroglobulin and the evolutionary relationship among these three proteins is discussed.     

Nucleotide sequence of the gene encoding the serotype-specific antigen of human (Wa) rotavirus: comparison with the homologous genes from simian SA11 and UK bovine rotaviruses

The nucleotide sequence of human (Wa) rotavirus genome segment 9, which encodes the serotype-specific antigen VP7, has been determined. Comparison of the deduced amino acid sequence of Wa VP7 protein to the sequences of simian SA11 and UK bovine VP7 proteins shows that the majority of the amino acid differences are clustered between amino acid residues 37 through 49, 65 through 75, 87 through 105, 122 through 126, 146 through 149, 178 through 181, and 208 through 242. A hydrophilicity profile of the three proteins reveals correlations between hydrophilic peaks, potentially antigenic determinants, and certain clusters of amino acid changes.     

Nucleotide sequence of rat liver aldolase B messenger RNA

The nucleotide sequence of messenger RNA encoding rat liver aldolase B has been determined by sequence analysis using recombinant cDNAs cloned in bacterial plasmids. The sequence contains part of the 5'-untranslatable region (68 nucleotides), the entire coding region (1092 nucleotides), and the complete 3'-untranslatable region (387 nucleotides), excluding the poly(A) tail. A potential ribosomal-binding site is located about 30 nucleotides upstream from the initiation codon. The amino acid sequence of rat liver aldolase B is composed of 364 amino acids and has 70% homology with rabbit muscle aldolase A.     

Generation of recombinant rotavirus with an antigenic mosaic of cross-reactive neutralization epitopes on VP4

Recombinant rotavirus (RV) with cDNA-derived chimeric VP4 was generated using recently developed reverse genetics for RV. The rescued virus, KU//rVP4(SA11)-II(DS-1), contains SA11 (simian RV strain, G3P[2])-based VP4, in which a cross-reactive neutralization epitope (amino acids 381 to 401) on VP5* is replaced by the corresponding sequence of a different P-type DS-1 (human RV strain, G2P[4]). Serological analyses with a panel of anti-VP4- and -VP7-neutralizing monoclonal antibodies revealed that the rescued virus carries a novel antigenic mosaic of cross-reactive neutralization epitopes on its VP4 surface. This is the first report of the generation of a recombinant RV with artificial amino acid substitutions.     

Isolation and analysis of a cDNA coding for human C1 inhibitor

A cDNA coding for C1 inhibitor was isolated from a human liver lambda gt11 expression library and sequenced by the dideoxy method. The amino acid sequence deduced from the cDNA indicated that the insert was a partial clone coding for 310 amino acids including the reactive site present at the carboxyl end of the molecule. The reactive site corresponds to that previously reported by Salvesen et al. (J. Biol. Chem. 260, 2432, 1985). The cDNA also contained a stop codon of TGA, 264 nucleotides at the 3' noncoding region, and a polyadenylation signal sequence of AATAAA 15 nucleotides upstream from the poly(A) tail. The amino acid sequence flanking the reactive site of the inhibitor is homologous to other members of the superfamily of plasma serine protease inhibitors.     

Serotypic analysis of VP3 and VP7 neutralization escape mutants of rhesus rotavirus.

Neutralization escape mutants of simian rotaviruses (rhesus rotavirus and SA11) were tested in hemagglutination inhibition and neutralization assays against hyperimmune and infection sera to determine if mutation in an immunodominant epitope could enable neutralization escape. An SA11 mutant with a new glycosylation site at amino acid 211 of VP7 was shown to escape neutralization by hyperimmune but not infection sera.     

Rotaviruses code for two types of glycoprotein precursors

Rotaviruses are nonenveloped viruses that code for two glycoproteins: a structural glycoprotein (VP7) and a nonstructural glycoprotein (NS29). The precursor to VP7 (37K) was shown to contain a 1.5K cleavable signal sequence. The 37K precursor was authentically processed (signal sequence cleaved and the polypeptide "core" glycosylated) when synthesized in a cell-free system supplemented with dog pancreatic microsomes. Similar experiments were performed with the nonstructural glycoprotein precursor (20K); however, the 20K precursor contained an integral (noncleavable) signal sequence. Both precursors were inserted into membranes cotranslationally and both glycosylated products underwent posttranslational oligosaccharide processing. The results suggest a morphogenetic scheme for the simian rotavirus SA11.     

人A组轮状病毒北京地方株VP4血清型特异片段编码基因的序列分析

轮状病毒是引起婴幼儿严重腹泻的重要病原,其第四基因编码主要中和抗原VP4,而VP4可裂解为VP8和VP5两个片段。VP8为抗原型特异性片段。克隆并测定了具有代表性的三个轮状病毒北京株VP4编码基因5′端（VPS＋VPS一部分）887个核苷酸序列并据此推导出其氨基酸序列。结果表明,相同血清型的地方株和标准株之间具有高度同源性（92％～966％）,不同血清型间则变异较大（70．5％～71％）。氨基酸最大变异处位于aa84～172,并对胰酶作用位点在致病性中的可能性进行了讨论。     

Localization of rotavirus VP4 neutralization epitopes involved in antibody-induced conformational changes of virus structure.

We previously characterized three neutralization-positive epitopes (NP1 [1a and 1b], NP2, and NP3) and three neutralization-negative epitopes on the simian rotavirus SA11 VP4 with 13 monoclonal antibodies (MAbs). Conformational changes occurred as a result of the binding of NP1 MAbs to the SA11 spike VP4, and enhanced binding of all neutralization-negative MAbs was observed when NP1 MAbs bound VP4 in a competitive MAb capture enzyme-linked immunosorbent assay. To further understand the structure and function of VP4, we have continued studies with these MAbs. Electron microscopic and sucrose gradient analyses of SA11-MAb complexes showed that triple-layered viral particles disassembled following treatment with NP1b MAbs 10G6 and 7G6 but not following treatment with NP1a MAb 9F6, NP2 MAb 2G4, and NP3 MAb 23. Virus infectivity was reduced approximately 3 to 5 logs by the NP1b MAbs. These results suggest that NP1b MAb neutralization occurs by a novel mechanism. We selected four neutralization escape mutants of SA11 with these VP4 MAbs and characterized them by using plaque reduction neutralization assays, hemagglutination inhibition assays, and an antigen capture enzyme-linked immunosorbent assay. These analyses support the previous assignment of the NP1a, NP1b, NP2, and NP3 MAbs into separate epitopes and confirmed that the viruses were truly neutralization escape mutants. Nucleotide sequence analyses found 1 amino acid (aa) substitution in VP8* of VP4 at (i) aa 136 for NP1a MAb mutant 9F6R, (ii) aa 180 and 183 for NP1b MAb mutants 7G6R and 10G6R, respectively, and (iii) aa 194 for NP3 MAb mutant 23R. The NP1b MAb mutants showed an unexpected enhanced binding with heterologous nonneutralization MAb to VP7 compared with parental SA11 and the other mutants. Taken together, these results suggest that the NP1b epitope is a critical site for VP4 and VP7 interactions and for virus stability.     

Amino acid sequence of human histidine-rich glycoprotein derived from the nucleotide sequence of its cDNA

A lambda gt 11 library containing cDNA inserts prepared from human liver mRNA has been screened with an affinity-purified antibody to human histidine-rich glycoprotein (HRG) and then with a restriction fragment isolated from the 5' end of the largest cDNA insert obtained by antibody screening. A number of positive clones were identified and shown to code for HRG by DNA sequence analysis. A total of 2067 nucleotides were determined by sequencing 3 overlapping cDNA clones, which included 121 nucleotides of 5'-noncoding sequence, 54 nucleotides coding for a leader sequence of 18 amino acids, 1521 nucleotides coding for the mature protein of 507 amino acids, a stop codon of TAA, and 352 nucleotides of 3'-noncoding sequence followed by a poly(A) tail of 16 nucleotides. The length of the noncoding sequence of the 3' end differed in several clones, but each contained a polyadenylylation or processing sequence of AATAAA followed by a poly(A) tail. More than half of the amino acid sequence of HRG consisted of five different types of internal repeats. Within the last 3 internal repeats (type V), there were 12 tandem repetitions of a 5 amino acid segment with a consensus sequence of Gly-His-His-Pro-His. This repeated portion, referred to as a "histidine-rich region", contained 53% histidine and showed a high degree of similarity to a histidine-rich region of high molecular weight kininogen.     

Nucleotide sequence of rat alpha 1-acid glycoprotein messenger RNA

The complete nucleotide sequence of rat alpha 1-acid glycoprotein (alpha 1-AGP) mRNA has been determined from cloned double-stranded cDNA. The coding portion of the mRNA was bounded at the ends by a 5'-untranslated region of 35 nucleotides in length and a 3'-untranslated region of 119 nucleotides in length. The 3'-untranslated region contains the characteristic AAUAAA sequence ending 18 nucleotides from the 3'-terminal poly(A) segment. The 5'-region of the mRNA contains two in-phase AUG codons separated by 12 nucleotides. Comparison with the known NH2-terminal amino acid sequence of serum rat alpha 1-AGP suggests that the primary translation product of the mRNA contains an additional 14 or 18 amino acids that are not present in the mature form of the protein, which contains 187 amino acids. The inferred amino acid sequence of rat alpha 1-AGP and the known amino acid sequence of human alpha 1-AGP have several regions of identity clustered in the NH2-terminal portion of the proteins. The carboxyl-terminal regions show significantly less homology. Six potential asparagine glycosylation sites are found in the rat sequence, and four of these sites are in positions similar to known glycosylation sites in the human protein. Furthermore, three of these potential glycosylation sites are in a region that exhibits extensive amino acid sequence conservation, suggesting that this region may be important for the biological function of alpha 1-AGP.     

Antibodies specific for the carboxy-terminal region of the major surface glycoprotein of simian rotavirus (SA11) and human rotavirus (Wa)

Antibodies specific for the major outer capsid protein (VP7) of the simian rotavirus SA11 were obtained by immunization of rabbits with a synthetic peptide, Ser-Ala-Ala-Phe-Tyr-Tyr-Arg-Val, corresponding to the eight carboxy-terminal amino acids of the viral protein predicted from the nucleotide sequence of the gene segment 9 of the SA11 genome. As the carboxy-terminal region of the VP7 of human rotavirus Wa has an identical sequence, cross-reactivity of the raised antibodies was observed with this strain.     

Cloning and sequence of UK bovine rotavirus gene segment 7: marked sequence homology with simian rotavirus gene segment 8.

The genome of the UK bovine rotavirus, which consists of eleven segments of dsRNA was polyadenylated and reverse-transcribed into cDNA. Complementary cDNA strands were annealed and the termini of the duplexes completed using DNA polymerase I. Full-length DNA copies of RNA segments 7, 8 and 9 were cloned into the Pst I site of pBR322 and a clone containing the entire gene 7 was identified and sequenced. Gene 7 is 1059 nucleotides in length and contains a single long open reading frame capable of coding for a protein of 317 amino-acids. The known gene product of segment 7 is a protein with an estimated molecular weight of 33,000 daltons. When the UK bovine rotavirus gene 7 sequence was compared with the published data for the homologous gene (segment 8) of the simian rotavirus SA11, it was found to be identical to it in size and the arrangement of the proposed coding and non-coding regions, and very similar in nucleotide sequence (88% homology). Most of the base changes are silent and the predicted amino-acid sequences are almost identical (96% homology).