Similarity in gene organization and homology between proteins of animal picornaviruses and a plant comovirus suggest common ancestry of these virus families

The amino acid sequences deduced from the nucleic acid sequences of several animal picornaviruses and cowpea mosaic virus (CPMV), a plant virus, were compared. Good homology was found between CPMV and the picornaviruses in the region of the picornavirus 2C (P2-X protein), VPg, 3C pro (proteinase) and 3D pol (RNA polymerase) regions. The CPMV B genome was found to have a similar gene organization to the picornaviruses. A comparison of the 3C pro (proteinase) regions of all of the available picornavirus sequences and CPMV allowed us to identify residues that are completely conserved; of these only two residues, Cys-147 and His-161 (poliovirus proteinase) could be the reactive residues of the active site of a proteinase with analogous mechanism to a known proteinase. We conclude that the proteinases encoded by these viruses are probably cysteine proteinases, mechanistically related, but not homologous to papain.     

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.     

Identification of a protease gene of human T-cell leukemia virus type I (HTLV-I) and its structural comparison

We determined the nucleotide sequence of a region between the gag and pol genes of a replication-competent proviral clone of a human T-cell leukemia virus type I (HTLV-I) from MT-2 cells. This region overlapping the gag and pol genes contains an open reading frame with a different phase from others. The deduced amino acid sequences show significant homology with the known protease gene of other retroviruses, and harbors highly conserved amino acid sequences that are well conserved in other retroviral protease domains. These results indicate that this open reading frame encodes a HTLV-I protease.     

Analysis of the complete nucleotide sequence of the picornavirus Theiler's murine encephalomyelitis virus indicates that it is closely related to cardioviruses

Theiler's murine encephalomyelitis viruses (TMEV) are naturally occurring enteric pathogens of mice which constitute a separate serological group within the picornavirus family. Persistent TMEV infection in mice provides a relevant experimental animal model for the human demyelinating disease multiple sclerosis. To provide information about the TMEV classification, genome organization, and protein processing map, we determined the complete nucleotide sequence of the TMEV genome and deduced the amino acid sequence of the polyprotein coding region. The RNA genome, which is typical of the picornavirus family, is 8,098 nucleotides long. The 5' untranslated region is 1,064 nucleotides long (making it the longest in the picornavirus family after the aphthoviruses) and lacks a poly(C) tract. Computer-generated comparison of the 5' and 3' noncoding regions and polyprotein revealed the highest level of nucleotide and predicted amino acid identity between the TMEV and the cardioviruses encephalomyocarditis virus (EMCV) and Mengo virus. The TMEV polyprotein, which appears to be processed like EMCV since the amino acids flanking the putative proteolytic cleavage sites have been conserved, begins with a short leader peptide followed by 11 other gene products in the standard L-4-3-4 picornavirus arrangement. Because of these similarities, we propose that the TMEV be grouped with the cardioviruses. However, since TMEV and EMCV have different biophysical properties and show no cross-neutralization, they most likely belong in a separate cardiovirus subgroup.     

Nucleotide sequence analysis of cDNA encoding the coat protein of cucumber mosaic virus: genome organization and molecular features of the protein

The cDNA sequence coding for the coat protein of cucumber mosaic virus (Japanese Y strain) was cloned, and its nucleotide sequence was determined. The sequence contains an open reading frame that encodes the coat protein composed of 218 amino acids. The nucleotide and deduced amino acid sequences of the coat protein of this strain were compared with those of the Q strain; the homologies of the sequences were 78% and 81%, respectively. Further study of the sequences gave an insight into the genome organization and the molecular features of the coat protein. The coding region can be divided into three characteristic regions. The N-terminal region has conserved features in the positively charged structure, the hydropathy pattern and the predicted secondary structure, although the amino acid sequence is varied mainly due to frameshift mutations. It is noteworthy that the positions of arginine residues in this region are highly conserved. Both the nucleotide and amino acid sequences of the central region are well conserved. The amino acid sequence of the C-terminal region is not conserved, because of frameshift mutations, however, the total number of amino acids is conserved. The nucleotide sequence of the 3'-noncoding region is divergent, but it could form a tRNA-like structure similar to those reported for other viruses. Detailed investigation suggests that the Y and Q strains are evolutionarily distant.     

Rat beta casein cDNA: sequence analysis and evolutionary comparisons.

The complete sequence of a 1072 nucleotide rat beta-casein cDNA insertion in the hybrid plasmid pC beta 23 has been determined. Primer extension was employed to determine the sequence of an additional 82 5'-terminal nucleotides in beta-casein mRNA. Rat beta-casein mRNA consists of a 696 nucleotide coding region, flanked by 52 nucleotide 5' and 406 nucleotide 3' noncoding regions, including a 40 nucleotide poly(A) tail. The derived 216 amino acid sequence of rat beta-casein was compared to the previously determined sequences of beta-caseins from several other species. Approximately 38% of the amino acids have been conserved among the rat, ovine, bovine and human sequences and these conserved amino acids occurred in clusters throughout the protein. One such cluster containing the majority of the potential casein phosphorylation sites was located near the amino terminus. Contrary to the considerable divergence observed for the processed beta-casein, 14 of 15 amino acids in the signal peptide sequence of the precasein were identical between the rat and ovine caseins.     

Sequence analysis of NS3 protease gene in clinical strains of hepatitis C virus

The amino terminal region of the non structural gene 3 (NS3) of hepatitis C virus (HCV) is a chymotripsinlike serine-protease responsible for cleavage of the non structural proteins of Hepatitis C virus (HCV). In order to investigate the genetic variation of this region, we developed a nested PCR to obtain NS3 protease sequences from 54 patients chronically infected with HCV genotypes 1a, 1b and 3, respectively. Comparison of nucleotide and amino acids sequences of NS3 protease domain with consensus sequence obtained within the same genotype, showed 3.73% nucleotide divergence and 1.64% amino acid divergence in isolates of genotype 3a, whereas isolates 1a exhibited 4.45% nucleotide and 4% amino acid change, respectively. Finally, NS3 sequence from 1b isolates revealed 6.47% nucleotide and 3.5 % aa changes. Comparison of consensus amino acid sequences derived from isolates 1a, 1b and 3, with the HCV prototypes showed a low amino acid sequence diversity. However, the consensus sequence of HCV genotype 3 isolates showed an amino acid changed from the prototype, that was located within a region important for enzyme structure and activity. These results indicated that the NS3 protease gene is highly conserved within the same HCV genotype. The domains involved in enzyme function were highly conserved in 1a and 1b strains, whereas consensus sequence of isolates 3a showed that the majority of these strains were not perfectly conserved in one of such regions. These findings altogether suggested that the NS3 protease enzyme of HCV may constitute an important target for antiviral therapy, but the NS3 protease variability of isolates 3 within a region that is a potential target for antiviral therapy could pose a problem for structure based drug development.     

Isolation and characterization of the rat proenkephalin gene

The rat proenkephalin gene has been isolated by molecular cloning and characterized by DNA-sequence analysis. The gene exhibits a structural organization similar to that of the human gene. The nucleotide sequence encoding the biologically active opioid peptides which are generated from the proenkephalin precursor as well as the 3' untranslated region of the mRNA are found on a large exon at the 3' end of the gene (Exon III). The nucleotide sequence encoding the N terminus of the mature protein and its signal peptide are located on Exon II while Exon I encodes the 5' untranslated region of the mRNA. The nucleotide sequence of these exons and their flanking regions has been determined and compared to the human proenkephalin gene. Analysis of the nucleotide sequence homology between the human and rat proenkephalin gene reveals the presence of highly conserved regions within both the coding and noncoding portions of the genes. Enkephalin-coding sequences as well as 5' flanking sequences appear to be the most highly conserved. The importance and possible function of these sequences are discussed.     

Structure of the gene encoding the circumsporozoite protein of Plasmodium yoelii. A rodent model for examining antimalarial sporozoite vaccines

The gene encoding the circumsporozoite protein (CSP) from the rodent malaria parasite, Plasmodium yoelii, has been cloned and the nucleotide sequence has been determined. The gene encodes a protein of 367 amino acids as deduced from the nucleotide sequence. This gene is structurally similar to other Plasmodium spp. CSP genes in that it contains putative hydrophobic signal and anchor sequences at the NH2 and COOH termini, respectively, two small regions (Regions I and II) that are conserved in all CSP genes analyzed to date, and a central region containing the immunodominant repeating peptide sequence. Unlike other CSP genes, however, the immunodominant repeat region of the gene is composed of two distinctly different types of tandem repeats. One repeating unit is six amino acids (Gln-Gly-Pro-Gly-Ala-Pro) in length while the other is only four (Gln-Gln-Pro-Pro) residues long. A synthetic peptide, Gln-Gly-Pro-Gly-Ala-Pro X 3, strongly inhibits the binding of anti-CSP monoclonal antibody to sporozoite antigens while another peptide, Gln-Gln-Pro-Pro X 4, weakly inhibits the binding of this same antibody to sporozoite antigens. This work should allow the construction of a mouse model system to parallel human vaccine trials.     

Conservation of human and mouse basonuclins as a guide to important features of the protein

The nucleotide sequence of mouse basonuclin has been determined from its cDNA by PCR and compared with the previously known sequence of human basonuclin. Overall, there is 88% identity in the encoded amino acid sequences, but some regions have been much more conserved than others. Zinc fingers 2 and 6, the region containing the nuclear localization signal and the region containing the serine stripe encode identical amino acid sequences in the two species, but differ by numerous silent nucleotide substitutions, suggesting that these regions are likely to be important for the functions of the protein common to the two species. Similarly, zinc fingers 1 and 5 diverge at only a single amino acid residue. In contrast, other regions of the sequence have diverged considerably, such as zinc fingers 3 and 4. The region adjacent to the N-terminus is very divergent and this aids in locating the translation start site. The highly conserved regions are likely to be essential for the common function of the proteins, and the more divergent regions may be either unconstrained or adapted to different requirements in the two species.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

Cloning and expression of a cDNA encoding the laccase from Schizophyllum commune

We cloned and analyzed the nucleotide sequence of a cDNA that encodes polyphenol oxidase (laccase) from the white-rot basidiomycete Schizophyllum commune. The nucleotide sequence of the full-length cDNA contained a 1554-base open reading frame that encoded a polypeptide of 518 amino acid residues, including a putative signal peptide of 16 residues. It contained four highly similar regions that are conserved in the deduced amino acid sequences of other laccases, including the region thought to be involved in copper binding. Aspergillus sojae strain 1860 (which has low protease levels) was transformed with the plasmid lacAL/pTPT, which contained the laccase gene under the control of the tannase promoter from Aspergillus oryzae. Laccase was secreted into the medium when transformants A1 and A2 were cultured in tannic acid-containing medium.     

Human DNA sequences homologous to a protein coding region conserved between homeotic genes of Drosophila

Several human DNA sequences were isolated by virtue of homology to a highly conserved region that has been identified in a number of homeotic genes in Drosophila. Structural analysis of the human DNAs indicate that two separate and distinct regions sharing a high degree of homology with the homeo box sequences of Drosophila are separated by only 5 kb in the human genome. Sequence determination of these regions reveals that both human DNA sequences contain a region capable of coding 61 amino acids, which shares greater than 90% homology with the peptide sequences specified by the homeo box domain of Drosophila homeotic genes, Antennapedia, fushi tarazu, and Ultrabithorax. By contrast, the human DNA sequences lying outside of the 190 nucleotide homeo box region share virtually no sequence homology, either with the flanking sequences of the other human clones or with flanking regions of the known Drosophila homeotic genes.     

Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters

A neutral protease with an estimated M_r of about 26 kD and responsible for cleavage of myosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced. Although the hamster protein shared 63-85% identity in nucleotide and amino acid sequences with rat and mouse mast cell proteases, it had a higher degree of specificity for myosin LC2 than mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin because of its unique enzymatic specificities to distinguish it from other mast cell proteases. A polyclonal antibody was raised specific to the hamster muscle and human cardiac muscle mekratins without apparent cross-reaction with rat mast cell proteases. We have earlier demonstrated the presence in excess of a neutral protease that specifically cleaves LC2 in human hearts obtained at end stage idiopathic dilated cardiomyopathy (IDC). Western analyses revealed that heart tissue from patients with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was similar to that seen in myopathic hamster skeletal muscle. No bands were recognized by the antibody when IDC myofibrils were probed due to the removal of soluble proteins during sample preparation. Thus, these results strongly suggest that the anti-mekratin antibody will provide positive identification of IDC in many cases and diagnosis by exclusion may be replaced.     

RNA-dependent RNA polymerase gene sequence from foot-and-mouth disease virus in Hong Kong

A foot-and-mouth disease virus (FMDV, HKN/2002) was isolated in Hong Kong in 2002. The nucleotide sequence of the 3D(pol) gene encoding the viral RNA-dependent RNA polymerase was determined and compared with that of the same gene from other FMDVs. The 3D(pol) gene was 1410 nucleotides in length encoding a protein of 470 amino acid residues. Sequence comparisons indicated that HKN/2002 belonged to serotype O. An evolutionary tree based on the 3D(pol) sequences of 20 FMDV isolates revealed that the nucleotide sequence of the HKN/2002 3D(pol) gene was most similar to those of isolates found in Taiwan in 1997, suggesting that they share a common ancestor. The amino acid sequence of the HKN/2002 3D(pol) gene was determined and aligned with those of representative isolates from seven other Picornaviridae genera. Eight highly conserved regions were detected, indicating a conserved functional relevance for these motifs. Alignment of 20 FMDV 3D(pol) amino acid sequences revealed a hypermutation region near the N-terminus that may help the virus evade host immune systems.     

Functional determinants of the Epstein-Barr virus protease

Herpesvirus proteases are essential for the production of progeny virus. They cleave the assembly protein that fills the immature capsid in order to make place for the viral DNA. The recombinant protease of the human gamma-herpesvirus Epstein-Barr virus (EBV) was expressed in Escherichia coli and purified. Circular dichroism indicated that the protein was properly folded with a secondary structure content similar to that of other herpesvirus proteases. Gel filtration and sedimentation analysis indicated a fast monomer-dimer equilibrium of the protease with a K(d) of about 60 microM. This value was not influenced by glycerol but was lowered to 1.7 microM in the presence of 0.5 M sodium citrate. We also developed an HPLC-based enzymatic assay using a 20 amino acid residue synthetic peptide substrate derived from one of the viral target sequences for the protease. We found that conditions that stabilised the dimer also led to a higher enzymatic activity. Through sequential deletion of amino acid residues from either side of the cleavage site, the minimal peptide substrate for the protease was determined as P5-P2'. This minimal sequence is shorter than that for other herpesvirus proteases. The implications of our findings are discussed with reference to the viral life-cycle. These results are the first ever published on the EBV protease and represent a first step towards the development of protease inhibitors.     

Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses.

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.     

The complete sequence of the mouse skeletal alpha-actin gene reveals several conserved and inverted repeat sequences outside of the protein-coding region.

The complete nucleotide sequence of a genomic clone encoding the mouse skeletal alpha-actin gene has been determined. This single-copy gene codes for a protein identical in primary sequence to the rabbit skeletal alpha-actin. It has a large intron in the 5'-untranslated region 12 nucleotides upstream from the initiator ATG and five small introns in the coding region at codons specifying amino acids 41/42, 150, 204, 267, and 327/328. These intron positions are identical to those for the corresponding genes of chickens and rats. Similar to other skeletal alpha-actin genes, the nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. Comparison of the nucleotide sequences of rat, mouse, chicken, and human skeletal muscle alpha-actin genes reveals conserved sequences (some not previously noted) outside of the protein-coding region. Furthermore, several inverted repeat sequences, partially within these conserved regions, have been identified. These sequences are not present in the vertebrate cytoskeletal beta-actin genes. The strong conservation of the inverted repeat sequences suggests that they may have a role in the tissue-specific expression of skeletal alpha-actin genes.     

The gene encoding DRAP (BACE2), a glycosylated transmembrane protein of the aspartic protease family, maps to the down critical region

We applied cDNA selection methods to a genomic clone (YAC 761B5) from chromosome 21 located in the so-called 'Down critical region' in 21q22.3. Starting from human fetal heart and brain mRNAs we obtained and sequenced several cDNA clones. One of these clones (Down region aspartic protease (DRAP), named also BACE2 according to the gene nomenclature) revealed a striking nucleotide and amino acid sequence identity with several motifs present in members of the aspartic protease family. In particular the amino acid sequences comprising the two catalytic sites found in all mammalian aspartic proteases are perfectly conserved. Interestingly, the predicted protein shows a typical membrane spanning region; this is at variance with most other known aspartic proteases, which are soluble molecules. We present preliminary evidence, on the basis of in vitro translation studies and cell transfection, that this gene encodes a glycosylated protein which localizes mainly intracellularly but to some extent also to the plasma membrane. Furthermore DRAP/BACE2 shares a high homology with a newly described beta-secretase enzyme (BACE-1) which is a transmembrane aspartic protease. The implications of this finding for Down syndrome are discussed.