Mapping of the Feline Calicivirus Proteinase Responsible for Autocatalytic Processing of the Nonstructural Polyprotein and Identification of a Stable Proteinase-Polymerase Precursor Protein

Expression of the region of the feline calicivirus (FCV) ORF1 encoded by nucleotides 3233 to 4054 in an in vitro rabbit reticulocyte system resulted in synthesis of an active proteinase that specifically processes the viral nonstructural polyprotein. Site-directed mutagenesis of the cysteine (Cys1193) residue in the putative active site of the proteinase abolished autocatalytic cleavage as well as cleavage of the viral capsid precursor, suggesting that this "3C-like" proteinase plays an important role in proteolytic processing during viral replication. Expression of the region encoding the C-terminal portion of the FCV ORF1 (amino acids 942 to 1761) in bacteria allowed direct N-terminal sequence analysis of the virus-specific polypeptides produced in this system. The results of these analyses indicate that the proteinase cleaves at amino acid residues E960-A961, E1071-S1072, E1345-T1346, and E1419-G1420; however, the cleavage efficiency is varied. The E1071-S1072 cleavage site defined the N terminus of a 692-amino-acid protein that contains sequences with similarity to the picornavirus 3C proteinase and 3D polymerase domains. Immunoprecipitation of radiolabeled proteins from FCV-infected feline kidney cells with serum raised against the FCV ORF1 C-terminal region showed that this "3CD-like" proteinase-polymerase precursor protein is apparently stable and accumulates in cells during infection.     

Processing map and essential cleavage sites of the nonstructural polyprotein encoded by ORF1 of the feline calicivirus genome

Feline calicivirus (FCV) nonstructural proteins are translated as part of a large polyprotein that undergoes autocatalytic processing by the virus-encoded 3C-like proteinase. In this study, we mapped three new cleavage sites (E(46)/A(47), E(331)/D(332), and E(685)/N(686)) recognized by the virus proteinase in the N-terminal part of the open reading frame 1 (ORF1) polyprotein to complete the processing map. Taken together with two sites we identified previously (E(960)/A(961) and E(1071)/S(1072)), the FCV ORF1 polyprotein contains five cleavage sites that define the borders of six proteins with calculated molecular masses of 5.6, 32, 38.9, 30.1, 12.7, and 75.7 kDa, which we designated p5.6, p32, p39 (NTPase), p30, p13 (VPg), and p76 (Pro-Pol), respectively. Mutagenesis of the E to A in each of these cleavage sites in an infectious FCV cDNA clone was lethal for the virus, indicating that these cleavages are essential in a productive virus infection. Mutagenesis of two cleavage sites (E(1345)/T(1346) and E(1419)/G(1420)) within the 75.7-kDa Pro-Pol protein previously mapped in bacterial expression studies was not lethal.     

Localization of the active site of the alpha subunit of the Escherichia coli DNA polymerase III holoenzyme.

Using a deletion approach on the alpha subunit of DNA polymerase III from Escherichia coli, we show that there is an N-proximal polymerase domain which is distinct from a more C-proximal tau and beta binding domain. Although deletion of 60 residues from the alpha N terminus abolishes polymerase activity, deletions of 48, 169, and 342 amino acids from the C terminus progressively impair its catalytic efficiency but preserve an active site. Deletion of 342 C-terminal residues reduces k(cat) 46-fold, increases the Km for gapped DNA 5.5-fold, and increases the Km for deoxynucleoside triphosphates (dNTPs) twofold. The 818-residue protein with polymerase activity displays typical Michaelis-Menten behavior, catalyzing a polymerase reaction that is saturable with substrate and linear with time. With the aid of newly acquired sequences of the polymerase III alpha subunit from a variety of organisms, candidates for two key aspartate residues in the active site are identified at amino acids 401 and 403 of the E. coli sequence by inspection of conserved acidic amino acids. The motif Pro-Asp-X-Asp, where X is a hydrophobic amino acid, is shown to be conserved among all known DnaE proteins, including those from Bacillaceae, cyanobacteria, Mycoplasma, and mycobacteria. The E. coli DnaE deletion protein with only the N-terminal 366 amino acids does not have polymerase activity, consistent with the proposed position of the active-site residues.     

Crystal structure of norwalk virus polymerase reveals the carboxyl terminus in the active site cleft

Norwalk virus is a major cause of acute gastroenteritis for which effective treatments are sorely lacking. To provide a basis for the rational design of novel antiviral agents, the main replication enzyme in Norwalk virus, the virally encoded RNA-dependent RNA polymerase (RdRP), has been expressed in an enzymatically active form, and its structure has been crystallographically determined both in the presence and absence of divalent metal cations. Although the overall fold of the enzyme is similar to that seen previously in the RdRP from rabbit hemorrhagic disease virus, the carboxyl terminus, surprisingly, is located in the active site cleft in five independent copies of the protein in three distinct crystal forms. The location of this carboxyl-terminal segment appears to interfere with the binding of double-stranded RNA in the active site cleft and may play a role in the initiation of RNA synthesis or mediate interactions with accessory replication proteins.     

Effect of deletions within the leader peptide of pre-ornithine transcarbamylase on mitochondrial import

The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N-terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15-19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import.     

Promotion of the GTP-liganded state of the Go alpha protein by deletion of the C terminus.

G proteins are active as long as GTP is bound to the alpha subunit. Activation ends when GTP is cleaved to GDP that then stays bound to the active site. Agonist-liganded receptors allow formation of the active state by decreasing the affinity of alpha subunits for GDP allowing exchange of GDP for GTP. Since receptors interact with the C terminus of the alpha subunits, we tested whether deletion of the C terminus could mimic activation by receptors. Three deletions and one point mutation at the C terminus of alpha o were engineered in alpha o cDNA by the polymerase chain reaction, transcribed into RNA, and translated in a rabbit reticulocyte lysate. The ability of in vitro synthesized protein to bind guanine nucleotide was inferred from analysis of native tryptic cleavage patterns, while the ability of the proteins to associate with beta gamma was measured by sucrose density gradient centrifugation. Deletion of 14 amino acids, alpha oD[341], from the C terminus causes a large decrease in GDP affinity, with little or no change in guanosine 5'-3-O-(thio)triphosphate affinity. When GTP is present, alpha oD[341] remains in the activated conformation because exchange of GTP for GDP is rapid. Deletion of 10 amino acids, alpha oD[345], lowers GDP affinity, but less dramatically than in alpha oD[341]. Deletion of 5 amino acids, alpha oD[350], or mutation of Arg-349 to proline alpha oR[349P] has no detectable effects on GDP affinity. Deletion of up to 10 amino acids from the C terminus does not prevent formation of alpha beta gamma heterotrimers. We propose that the C terminus of the alpha subunit is a mobile region that blocks dissociation of GDP. Agonist-liganded receptors may move it aside to allow release of GDP, exchange for GTP, and activation of the alpha subunit.     

Structure-function studies of the herpes simplex virus type 1 DNA polymerase.

The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity.     

Truncation of NH2-terminal amino acid residues increases agonistic potency of leukotactin-1 on CC chemokine receptors 1 and 3

Leukotactin-1 (Lkn-1) is a human CC chemokine that binds to both CC chemokine receptor 1 (CCR1) and CCR3. Structurally, Lkn-1 is distinct from other human CC chemokines in that it has long amino acid residues preceding the first cysteine at the NH(2) terminus, and contains two extra cysteines. NH(2)-terminal amino acids of Lkn-1 were deleted serially, and the effects of each deletion were investigated. In CCR1-expressing cells, serial deletion up to 20 amino acids (Delta20) did not change the calcium flux-inducing activity significantly. Deletion of 24 amino acids (Delta24), however, increased the agonistic potency approximately 100-fold. Deletion of 27 or 28 amino acids also increased the agonistic potency to the same level shown by Delta24. Deletion of 29 amino acids, however, abolished the agonistic activity almost completely showing that at least 3 amino acid residues preceding the first cysteine at the NH(2) terminus are essential for the biological activity of Lkn-1. Loss of agonistic activity was due to impaired binding to CCR1. In CCR3-expressing cells, Delta24 was the only form of Lkn-1 mutants that revealed increased agonistic potency. Our results indicate that posttranslational modification is a potential mechanism for the regulation of biological activity of Lkn-1.     

The highly processive DNA polymerase of bacteriophage T5. Role of the unique N and C termini

The DNA polymerase encoded by bacteriophage T5 has been reported previously to be processive and to catalyze extensive strand displacement synthesis. The enzyme, purified from phage-infected cells, did not require accessory proteins for these activities. Although T5 DNA polymerase shares extensive sequence homology with Escherichia coli DNA polymerase I and T7 DNA polymerase, it contains unique regions of 130 and 71 residues at its N and C termini, respectively. We cloned the gene encoding wild-type T5 DNA polymerase and characterized the overproduced protein. We also examined the effect of N- and C-terminal deletions on processivity and strand displacement synthesis. T5 DNA polymerase lacking its N-terminal 30 residues resembled the wild-type enzyme albeit with a 2-fold reduction in polymerase activity. Deletion of 24 residues at the C terminus resulted in a 30-fold reduction in polymerase activity on primed circular DNA, had dramatically reduced processivity, and was unable to carry out strand displacement synthesis. Deletion of 63 residues at the C terminus resulted in a 20,000-fold reduction in polymerase activity. The 3' to 5' double-stranded DNA exonuclease activity associated with T5 DNA polymerase was reduced by a factor of 5 in the polymerase truncated at the N terminus but was stimulated by a factor of 7 in the polymerase truncated at the C terminus. We propose a model in which the C terminus increases the affinity of the DNA for the polymerase active site, thus increasing processivity and decreasing the accessibility of the DNA to the exonuclease active site.     

Isolation of an RNA-directed RNA polymerase-specific cDNA clone from tomato.

A 3600-bp RNA-directed RNA polymerase (RdRP)-specific cDNA comprising an open reading frame (ORF) of 1114 amino acids was isolated from tomato. The putative protein encoded by this ORF does not share homology with any characterized proteins. Antibodies that were raised against synthetic peptides whose sequences have been deduced from the ORF were shown to specifically detect the 127-kD tomato RdRP protein. The immunoresponse to the antibodies correlated with the enzymatic activity profile of the RdRP after chromatography on Q-, poly(A)-, and poly(U)-Sepharose, hydroxyapatite, and Sephadex G-200 columns. DNA gel blot analysis revealed a single copy of the RdRP gene in tomato. RdRP homologs from petunia, Arabidopsis, tobacco, and wheat were identified by using polymerase chain reaction. A sequence comparison indicated that sequences homologous to RdRP are also present in the yeast Schizosaccharomyces pombe and in the nematode Caenorhabditis elegans. The previously described induction of RdRP activity upon viroid infection is shown to be correlated with an increased steady state level of the corresponding mRNA. The possible involvement of this heretofore functionally elusive plant RNA polymerase in homology-dependent gene silencing is discussed.     

A recombinant form of Pseudomonas exotoxin directed at the epidermal growth factor receptor that is cytotoxic without requiring proteolytic processing.

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.     

High-level expression of a mitochondrial enzyme, ornithine transcarbamylase from rat liver, in a baculovirus expression system

The mitochondrial enzyme, ornithine transcarbamylase (OTC) from rat liver was expressed in Spodoptera frugiperda (Sf) insect cells using a baculovirus vector. When insect cells were infected with recombinant Autographica californica nuclear polyhedrosis virus (AcNPV) containing a cDNA encoding the precursor form of OTC (pOTC) inserted into the polyhedrin gene, they expressed catalytically active enzyme at levels of approximately 2.5 micrograms/10(6) cells. About 25% of the active enzyme was a novel, partially processed product of pOTC containing four extra amino acids at the amino terminus of OTC. The most abundant protein found in mitochondria from infected insect cells was the normal processing intermediate iOTC, which contains 8 extra amino acids at the amino terminus of OTC. Whereas this species, present at 20 micrograms/10(6) cells, was not active and did not bind the transition-state analog inhibitor of OTC, delta-PALO, the novel processing product did bind and was affinity-purified, along with mature OTC, on a PALO-affinity column. The OTC expressed in insect cells was located in the same compartment of the mitochondrion as in rat liver. The incomplete processing occurred in vitro in both noninfected and infected insect cells. The high level of expression of iOTC using the baculoviral expression system provides a means of overproducing an obligatory intermediate in the mitochondrial import process.     

Influenza virus polymerase basic protein 1 interacts with influenza virus polymerase basic protein 2 at multiple sites.

Three polymerase proteins of influenza type A virus interact with each other to form the active polymerase complex. Polymerase basic protein 1 (PB1) can interact with PB2 in the presence or absence of polymerase acidic protein. In this study, we investigated the domains of PB1 involved in complex formation with PB2 in vivo, using coexpression and coimmunoprecipitation of the PB1-PB2 complex with monospecific antibodies. Results show that PB1 possesses at least two regions which can interact independently and form stable complexes with PB2. Both of these regions are located at the NH2 terminus of PB1; the COOH-terminal half of PB1 is not involved in interacting with PB2. Deletion analysis further demonstrated that the interacting regions of PB1 encompass amino acids (aa) 48 to 145 and aa 251 to 321. Linker insertions throughout the PB1 sequences did not affect complex formation with PB2. Deletion and linker-insertion mutants of PB1 were tested for polymerase activity in vivo. For this analysis, we developed a simplified assay for viral polymerase activity that uses a reporter chloramphenicol acetyltransferase gene containing the 5' and 3' ends of influenza viral promoter and nontranslating regions (minus sense) of the NS gene joined to a hepatitis delta virus ribozyme at its 3' end. This assay demonstrated that all deletion mutants of PB1 exhibited either background or greatly reduced polymerase activity irrespective of the ability to interact with PB2 and that all linker-insertion mutants except one at the extreme COOH end (L-746) of PB1 were also negative for viral polymerase activity. These results show that compared with complex formation of PB1 with PB2, the polymerase activity of PB1 was extremely sensitive to structural perturbation.     

Mutational Analysis of Hepatitis C Virus NS5B in the Subgenomic Replicon Cell Culture

The hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme of HCV RNA replication. We previously identified five novel residues of NS5B in a JK-1 isolate indispensable for RdRP activity in vitro (Qin, W., Yamashita, T., Shirota, Y., Lin, Y., Wei, W., and Murakami, S. (2001) Hepatology 33, 728-737). We addressed the role of these residues in HCV RNA replication using a HCV replicon system derived from an M1LE isolate (Kishine, H., Sugiyama, K., Hijikata, M., Kato, N., Takahashi, H., Noshi, T., Nio, Y., Hosaka, M., Miyanari, Y., and Shimotohno, K. (2002) Biochem. Biophys. Res. Commun. 293, 993-999). The five residues of NS5B in M1LE were found to be critical for HCV replication in vivo and also indispensable for RdRP activity in vitro along with purified bacterial recombinant proteins. We also found a chimeric replicon of JK-1 and M1LE in which only the NS5B sequence derived from JK-1 could not replicate in Huh-7 cells. The residues responsible for the phenomenon were mapped by several chimeric and substituted forms of NS5B M1LE and/or JK-1 isolates in the HCV RNA replicon. Two residues, amino acids 220 and 288, were critical, and two residues, amino acids 213 and 231, were important for efficient HCV replication. Mutant JK-1 NS5B harboring all four residues of M1LE was replication-competent in the chimeric replicon and was as efficient as the original M1LE replicon. By comparing the replication competence in vivo and RdRP activity in vitro with various chimeric and mutated versions of NS5B, the HCV replication ability was found to correlate well with the RdRP activity. However, heat- and dilution-sensitive NS5Bs exhibiting weaker RdRP activity in vitro were found to be replication-incompetent, suggesting that HCV replication requires RdRP activity higher than a certain critical threshold.     

Taq DNA聚合酶功能区域的定位

通过参Ｕ法定点突变产生了ＴａｑＤＮＡ聚合酶Ｎ端分别缺失３个,２３５个,２８７个和４４３个氨基酸的４个缺失体,利用Ｂａｌ－３１连续缺失法产生了ＴａｑＤＮＡ聚合酶的Ｃ端分别缺失了２个、１６个、２９个、３２个、３４个氨基酸的５个缺失体．经ＤＮＡ聚合酶活性测定表明Ｎ端缺失３个,２３５个,２８７个氨基酸后活力和完整的Ｔａｑ相近,而缺失４４３个氨基酸后则失去了ＤＮＡ聚合酶活力;Ｃ端的５个缺失体都失去了ＤＮＡ聚合酶活性．据此ＴａｑＤＮＡ聚合酶的功能区域被定位在２８７～８３２氨基酸之间．     

Processing of yeast exoglucanase (beta-glucosidase) in a KEX2-dependent manner

We have detected proteolytic processing of a form of exoglucanase representative of the endoplasmic reticulum (form A). This processing did not take place when form A was obtained from protoplasts lysed in the presence of either EDTA or leupeptin, two wel-characterized inhibitors of KEX2 endoprotease from Saccharomyces cerevisiae. Sequencing of the amino terminus of an A-like form of enzyme secreted by a kex2 mutant indicated the presence of 4 amino acids, with a pair of basic residues (Lys-Arg) at their carboxyl side, preceding the amino terminus of the wild-type external exoglucanase.     

Direct interaction between nucleolin and hepatitis C virus NS5B

Hepatitis C virus (HCV) NS5B is an RNA-dependent RNA polymerase (RdRP), a central catalytic enzyme in HCV replication. While studying the subcellular localization of a NS5B mutant lacking the C-terminal membrane-anchoring domain, NS5Bt, we found that expression of the green fluorescent protein (GFP)-fused form was exclusively nucleolar. Interestingly, the distribution of endogenous nucleolin changed greatly in the cells expressing GFP-NS5B, with nucleolin colocalized with GFP-NS5B in perinuclear regions in addition to the nucleolus, suggesting that NS5B retains the ability to bind nucleolin. The interaction between nucleolin and NS5B was demonstrated by GST pull-down assay. GST pull-down assay results indicated that C-terminal region of nucleolin was important for its binding to NS5B. Scanning clustered alanine substitution mutants library of NS5B revealed two sites on NS5B that binds nucleolin. NS5B amino acids 208-214 and 500-506 were both found to be indispensable for the nucleolin binding. We reported that the latter sequence is essential for oligomerization of NS5B, which is a prerequisite for the RdRP activity. C-terminal nucleolin inhibited the NS5B RdRP activity in a dose-dependent manner. Taken together, this indicates the binding ability of nucleolin may be involved in NS5B functions.     

Autoproteolysis of the Extracellular Serine Proteinase of Lactococcus lactis subsp. cremoris Wg2

The molecular masses of purified extracellular serine proteinase of a number of Lactococcus lactis strains vary significantly, and these molecular mass values do not correspond to the values estimated on the basis of genetic data. The discrepancies can only partially be explained by N-terminal processing during maturation of the precursor enzyme and by C-terminal cleaving during the release from the cell envelope. With a monoclonal antibody that binds in the active site region of the L. lactis proteinase, the processing of the released proteinase was followed. At 30°C the proteinase was degraded with a concomitant loss of β-casein hydrolytic activity. In the presence of CaCl₂, proteinase degradation was inhibited, and new degradation products were detected. The specific serine proteinase inhibitors phenylmethylsulfonyl fluoride and diisopropylfluorophosphate also inhibited proteinase degradation. Two major high-molecular-mass proteinase fragments (165 and 90 kDa) were found to have the same N-terminal amino acid sequence as the mature proteinase, i.e., [Asp-1-Ala-2-Lys-3-Ala-4-Asn-5-Ser-6, indicating that both fragments were formed by cleavage at the C terminus. The N terminus of a proteinase fragment with low molecular mass (58 kDa) started with Gln-215. In this fragment part of the active site region was eliminated, suggesting that it is proteolytically inactive. Unlike larger fragments, this 58-kDa fragment remained intact after prolonged incubations. These results indicate that autoproteolysis of the L. lactis subsp. cremoris Wg2 proteinase ultimately leads to inactivation of the proteinase by deletion of the active site region.