Three-dimensional structure of aspartyl protease from human immunodeficiency virus HIV-1

The crystal structure of the protease of the human immunodeficiency virus type (HIV-1), which releases structural proteins and enzymes from viral polyprotein products, has been determined to 3 A resolution. Large regions of the protease dimer, including the active site, have structural homology to the family of microbial aspartyl proteases. The structure suggests a mechanism for the autoproteolytic release of protease and a role in the control of virus maturation.     

Cleavage of pre-tRNAs by the splicing endonuclease requires a composite active site

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5' and 3' splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively. A recent structure of an archaeal endonuclease complexed with a bulge-helix-bulge RNA has led to the unexpected hypothesis that catalysis requires a critical 'cation-pi sandwich' composed of two arginine residues that serve to position the RNA substrate within the active site. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5' splice site requires the conserved cation-pi sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.     

A structural model for the retroviral proteases

In many retroviruses the 5' end of the pol gene codes for a protease vital for the processing of the gag polyprotein into the separate core proteins. In some viruses this protease is encoded at the 3' end of the gag gene, or between the gag and pol genes in a different reading frame to either. A sequence, Asp-Thr-Gly, which is conserved in retroviral proteases is also conserved in the active sites of aspartic proteases, an observation which has led to the suggestion that the retroviral proteases could belong to this family. We have examined the sequences of the aspartic and retroviral protease families, using pattern-recognition, structure prediction and molecular modelling techniques, and conclude that the viral protease sequences probably correspond to a single domain of an aspartic protease and may function in a dimeric form. We have constructed a model of the pol-protease of human immunodeficiency virus 1 (HIV-1) to test this hypothesis.     

丙型肝炎病毒非结构蛋白NS3/4A的研究进展

丙型肝炎病毒(HCV)非结构蛋白NS3共有631个氨基酸组成,具有丝氨酸蛋白酶、三磷酸核苷酶(NTpase)和螺旋酶(Helicase)的功能,在HCV多聚蛋白的成熟和病毒复制过程中发挥着重要作用.非结构蛋白NS4A,其主要功能就是作为NS3丝氨酸蛋白酶的辅助因子,在HCV多氨酸成熟过程中发挥着不可替代的调节作用.此外,NS3/4A还参与NS5A的超磷酸化修饰过程.     

Inhibition of furin-mediated cleavage activation of HIV-1 glycoprotein gp160.

The envelope glycoprotein of human immunodeficiency virus (HIV) initiates infection by mediating fusion of the viral envelope with the cell membrane. Fusion activity requires proteolytic cleavage of the gp160 protein into gp120 and gp41 at a site containing several arginine and lysine residues. Activation at basic cleavage sites is observed with many membrane proteins of cellular and viral origin. We have recently found that the enzyme activating the haemagglutinin of fowl plague virus (FPV), an avian influenza virus, is furin. Furin, a subtilisin-like eukaryotic endoprotease, has a substrate specificity for the consensus amino-acid sequence Arg-X-Lys/Arg-Arg at the cleavage site. We show here that the glycoprotein of HIV-1, which has the same protease recognition motif as the FPV haemagglutinin, is also activated by furin.     

Searching for more effective HCV NS3 protease inhibitors via modification of corilagin

Corilagin was seperated from extract of Phyllanthus urinaria L. and used as the precursor of inhibitors of hepatitis C virus (HCV) NS3 serine protease. Six derivatives were obtained through the chemical modification of corilagin and their structures were elucidated by the spectra analysis. Bioassay of these compounds showed that two of them had improved inhibitory efficiency than the precursor, with IC₅₀ values of 2.28 μmol/L and 1.52 μmol/L, respectively. The binding mode of two active compounds with substrate binding site of HCV NS3 protease was also investigated by molecular docking method.     

Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease

HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation. The mature protease is active only as a dimer with each subunit contributing catalytic residues. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer, concomitant with the appearance of mature-like catalytic activity. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit beta-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species, we show that the mini-precursor forms highly transient, lowly populated (3-5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.     

Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase

Hepatitis C virus (HCV) is a human pathogen affecting nearly 3% of the world's population. Chronic infections can lead to cirrhosis and liver cancer. The RNA replication machine of HCV is a multi-subunit membrane-associated complex. The non-structural protein NS5A is an active component of HCV replicase, as well as a pivotal regulator of replication and a modulator of cellular processes ranging from innate immunity to dysregulated cell growth. NS5A is a large phosphoprotein (56-58 kDa) with an amphipathic alpha-helix at its amino terminus that promotes membrane association. After this helix region, NS5A is organized into three domains. The N-terminal domain (domain I) coordinates a single zinc atom per protein molecule. Mutations disrupting either the membrane anchor or zinc binding of NS5A are lethal for RNA replication. However, probing the role of NS5A in replication has been hampered by a lack of structural information about this multifunctional protein. Here we report the structure of NS5A domain I at 2.5-A resolution, which contains a novel fold, a new zinc-coordination motif and a disulphide bond. We use molecular surface analysis to suggest the location of protein-, RNA- and membrane-interaction sites.     

Complete mutagenesis of the HIV-1 protease

Retroviruses encode a protease which needs to be active for the production of infectious virions. A disabling mutation in the protease results in the production of non-infectious virus particles and examination of proteins from these mutant virions reveals unprocessed Gag and Gag-Pol precursor proteins, the substrates of the viral protease. Each amino acid of the HIV-1 protease was individually mutated using a simple mutagenesis procedure which is capable of introducing and identifying missense mutations in each residue of a protein. Phenotypic screening of these mutants in a heterologous assay system reveals three regions within the protease where multiple consecutive amino-acid residues are sensitive to mutation. These results show that random mutagenesis can be used to identify functionally important regions within a protein. Mutants with conditional phenotypes have also been identified within this collection.     

An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus

Hepatitis C virus (HCV) infection is a serious cause of chronic liver disease worldwide with more than 170 million infected individuals at risk of developing significant morbidity and mortality. Current interferon-based therapies are suboptimal especially in patients infected with HCV genotype 1, and they are poorly tolerated, highlighting the unmet medical need for new therapeutics. The HCV-encoded NS3 protease is essential for viral replication and has long been considered an attractive target for therapeutic intervention in HCV-infected patients. Here we identify a class of specific and potent NS3 protease inhibitors and report the evaluation of BILN 2061, a small molecule inhibitor biologically available through oral ingestion and the first of its class in human trials. Administration of BILN 2061 to patients infected with HCV genotype 1 for 2 days resulted in an impressive reduction of HCV RNA plasma levels, and established proof-of-concept in humans for an HCV NS3 protease inhibitor. Our results further illustrate the potential of the viral-enzyme-targeted drug discovery approach for the development of new HCV therapeutics.     

Antiviral compounds and one new iridoid glycoside from Cornus officinalis

From Cornus officinalis Sieb. et Zucc., bioassay-guided fractionation led to the isolation of four active tannin compounds with high effectiveness of inhibiting Hepatitis C virus NS3 serine protease in vitro. The compounds are: 1,2,3,6-tetragalloyl-β-D-glucopyranose (1), 1,2,3,4,6- pentagalloyl-β-D-glucopyranose (2), Tellimagrandin Ⅰ (3) and Tellimagrandin Ⅱ (4). The four compounds could inhibit HCV NS3 protease in vitro with IC50 values of 6.98, 5.11, 7.0 and 4.8 μmol/L respectively. In addition, a new iridoid glycoside (5) was also isolated from Cornus officinalis Sieb. et Zucc., which was assigned to be 7-O-butyl morroniside by spectroscopic analysis.     

Snapshots of tRNA sulphuration via an adenylated intermediate

Uridine at the first anticodon position (U34) of glutamate, lysine and glutamine transfer RNAs is universally modified by thiouridylase into 2-thiouridine (s2U34), which is crucial for precise translation by restricting codon-anticodon wobble during protein synthesis on the ribosome. However, it remains unclear how the enzyme incorporates reactive sulphur into the correct position of the uridine base. Here we present the crystal structures of the MnmA thiouridylase-tRNA complex in three discrete forms, which provide snapshots of the sequential chemical reactions during RNA sulphuration. On enzyme activation, an alpha-helix overhanging the active site is restructured into an idiosyncratic beta-hairpin-containing loop, which packs the flipped-out U34 deeply into the catalytic pocket and triggers the activation of the catalytic cysteine residues. The adenylated RNA intermediate is trapped. Thus, the active closed-conformation of the complex ensures accurate sulphur incorporation into the activated uridine carbon by forming a catalytic chamber to prevent solvent from accessing the catalytic site. The structures of the complex with glutamate tRNA further reveal how MnmA specifically recognizes its three different tRNA substrates. These findings provide the structural basis for a general mechanism whereby an enzyme incorporates a reactive atom at a precise position in a biological molecule.     

Structural basis for the bifunctionality of fructose-1,6-bisphosphate aldolase/phosphatase

Enzymes catalyse specific reactions and are essential for maintaining life. Although some are referred to as being bifunctional, they consist of either two distinct catalytic domains or a single domain that displays promiscuous substrate specificity. Thus, one enzyme active site is generally responsible for one biochemical reaction. In contrast to this conventional concept, archaeal fructose-1,6-bisphosphate (FBP) aldolase/phosphatase (FBPA/P) consists of a single catalytic domain, but catalyses two chemically distinct reactions of gluconeogenesis: (1) the reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GA3P) to FBP; (2) the dephosphorylation of FBP to fructose-6-phosphate (F6P). Thus, FBPA/P is fundamentally different from ordinary enzymes whose active sites are responsible for a specific reaction. However, the molecular mechanism by which FBPA/P achieves its unusual bifunctionality remains unknown. Here we report the crystal structure of FBPA/P at 1.5-? resolution in the aldolase form, where a critical lysine residue forms a Schiff base with DHAP. A structural comparison of the aldolase form with a previously determined phosphatase form revealed a dramatic conformational change in the active site, demonstrating that FBPA/P metamorphoses its active-site architecture to exhibit dual activities. Thus, our findings expand the conventional concept that one enzyme catalyses one biochemical reaction.     

Crystallographic studies on the activity of glycogen phosphorylase b

High resolution studies on the crystal structure of glycogen phosphorylase b have identified the catalytic site to which the substrate glucose-1-phosphate binds strongly with some local conformational changes. The site is situated 8 A (phosphate-to-phosphate distance) from pyridoxal phosphate, an essential cofactor of all glycogen phosphorylases. The catalytic site is 33 A from the site in the N-terminal portion of the molecule to which adenine nucleotides bind. In contrast to phosphorylase a (the active form of the enzyme which is phosphorylated at Ser 14), the positions of the first 19 residues of phosphorylase b are not well defined.     

Molecular characterization of H1N1 influenza A viruses from human cases in North America

Subtypes of H1N1 influenza virus can be found in humans in North America, while they are also associated with the infection of swine. Characterization of the genotypes of viral strains in human populations is important to understand the source and distribution of viral strains. Genomic and protein sequences of 10 isolates of the 2009 outbreak of influenza A （H1N1） virus in North America were obtained from GenBank database. To characterize the genotypes of these viruses, phylogenetic trees of genes PB2, PB1, PA, HA, NP, NA, NS and M were constructed by Phylip3.67 program and N-Linked glycosylation sites of HA, NA, PB2, NS1 and M2 proteins were analyzed online by NetNGlyc1.0 program. Phylogenetic analysis indicated that these isolates are virtually identical but may be recombinant viruses because their genomic fragments come from different viruses. The isolates also contain a characteristic lowly pathogenic amino acid motif at their HA cleavage sites （IPSIQSR↓GL）, and an E residue at position 627 of the PB2 protein which shows its high affinity to humans. The homologous model of M proteins showed that the viruses had obtained the ability of anti-amantadine due to the mutation at the drug-sensitive site, while sequence analysis of NA proteins indicated that the viruses are still susceptible to the neuraminidase inhibitor drug （i.e. oseltamivir and zanamivir） because no mutations have been observed. Our results strongly suggested that the viruses responsible for the 2009 outbreaks of influenza A （H1N1） virus have the ability to cross species barriers to infect human and mammalian animals based on molecular analysis. These findings may further facilitate the therapy and prevention of possible transmission from North America to other countries.     

利用质谱技术鉴定寨卡病毒非结构蛋白NS1的细胞内相互作用蛋白

寨卡病毒(Zika Virus)属于黄病毒科中的黄病毒属,虽然很早就已经被人类所发现,但是一直到2015年在南美巴西的大规模爆发,才引起了广泛的关注.寨卡病毒对人类的感染往往引起包括小头畸形和格林-巴利综合征在内的多种症状.寨卡病毒的基因组为单链正链RNA,其基因组可以编码翻译并剪切加工出3个结构蛋白,分别为膜蛋白,囊膜蛋白和核衣壳蛋白,以及7个非结构蛋白(NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5).相关研究已经证明,NS1蛋白与同属黄病毒属的登革病毒的发病有紧密的联系,而且根据其蛋白结构推测其可能与寨卡病毒穿越血脑屏障有关.因此鉴别NS1与细胞内的相互作用蛋白对于发现寨卡病毒在细胞内的转运,转录,以及装配都有重大的意义.在此,该课题构建并在HEK293细胞中表达包含Flag和Strep两种标签的NS1融合蛋白,通过免疫沉淀的方法将与NS1结合的蛋白利用标签蛋白进行分离,利用高分辨生物质谱技术,对蛋白进行分析鉴定.通过分别带有Flag与Strep标签的相互作用蛋白分析,发现了16个两种标签共同的结合蛋白,其进一步的通路分析证明这些蛋白于与病毒转录、病毒复制和免疫反应多个通路有关,相关的研究结果为今后进一步研究寨卡病毒的复制机制以及开发抗病毒药物提供了重要的参考价值.     

Nucleotide sequence of RNA2 and polyprotein processing sites of a Chinese isolate of broad bean wilt virus 2

RNA2 of broad bean wilt virus 2 (BBWV2) isolate B935 is composed of 3601 nucleotide (nt) residues, exclusive of the polyadenylate at the 3' end. Only one of the six possible reading frames has a long open reading frame, which extends from nt 231 to nt 3428 in the polarity of encapsidated RNA, and encodes a polyprotein of 119 kD. The N-terminus of the large coat protein (LCP) is located at 599 nt and the small coat protein (SCP) at 197 nl from the C-terminus of the 119 kD protein, which suggests that the coat proteins are released from the polyprotein by cleavages of a glulamine-glycine (Q-G) and a glutamine-alanine (Q-A) bond respectively. The sequence comparison of B935 with fabaviruses shows that B935 has very high sequence homology with other BBWV2 isolates and with patchoul' mild mosaic virus, but has lower homology with BBWV1 isolates. B935 has a similar genomic organization, but a low sequence homology to RNA2 molecules of comoviruses and nepoviruses.