马传染性贫血病病毒结构模型的建立

通过电子显微镜多种技术的观察和研究,明确了马传染性贫血病病毒的典型形态结构,即呈球形,表面附有顶端带钮状物的纤突,病毒膜为三层(单位膜),病毒基质充填于病毒膜和核芯亮之间,核芯壳呈园锥形,核芯为螺旋结构。在此基础上建立了马传染性贫血病病毒的结构模型。这是国内外首次报道,为深入研究该病毒和其有关病毒的特性奠定了基础,并为该病毒的分类提供了形态学依据。     

Mac1, a fission yeast transmembrane protein localizing to the poles and septum,is required for correct cell separation at high temperatures

Schizosaccharomyces pombe represents a genetic model system for studying cell polarity and division in eukaryotes. We report here the identification of Mac1, a novel fission yeast protein that localized predominantly to the cell tips and septum. Sequences corresponding to roughly the first 180 amino acids of Mac1, which exhibited weak homology to the transmembrane domains of the Aspergillus Pall protein [Mol. Microbiol. 30 (1998) 259], were found to specify localization to the cell periphery. The other 574 amino acids of Mac1 localized to the cytoplasm when expressed alone, thus suggesting that the N-terminal part of Mac1 functions as a plasma membrane anchor for the rest of the protein. In pom1 null mutant cells, which never switch from unipolar to bipolar growth but, instead, grow exclusively at the randomly chosen end [Genes Dev. 12 (1998) 1356], Mac1 was, nevertheless, found at both poles, thus suggesting that Mac1 does not specifically localize to the sites of growth. mac1 null mutant cells had no overt phenotype at 22-32 degrees C, but, nevertheless, displayed a marked decrease in viability at 34-36 degrees C, accompanied by severe separation defects. Overexpression of mac1 resulted in similar defects. Our data suggest that a correct dosage of Mac1 is needed for correct cell separation at elevated temperatures of growth.     

Influenza Hemagglutinin and Neuraminidase Membrane Glycoproteins

Considerable progress has been made toward understanding the structural basis of the interaction of the two major surface glycoproteins of influenza A virus with their common ligand/substrate: carbohydrate chains terminating in sialic acid. The specificity of virus attachment to target cells is mediated by hemagglutinin, which acquires characteristic changes in its receptor-binding site to switch its host from avian species to humans. Anti-influenza drugs mimic the natural sialic acid substrate of the virus neuraminidase enzyme but utilize the much tighter binding of the drugs for efficacy. Resistance to one of the two main antiviral drugs is differentially acquired by the two distinct subsets of neuraminidase as a consequence of structural differences in the enzyme active site between the two phylogenetic groups.     

Structure–function analysis of hepatitis C virus envelope glycoproteins E1 and E2

Hepatitis C virus (HCV) is the leading cause of chronic liver disease in humans. The envelope proteins of HCV are potential candidates for vaccine development. The absence of three-dimensional (3D) structures for the functional domain of HCV envelope proteins [E1.E2] monomer complex has hindered overall understanding of the virus infection, and also structure-based drug design initiatives. In this study, we report a 3D model containing both E1 and E2 proteins of HCV using the recently published structure of the core domain of HCV E2 and the functional part of E1, and investigate immunogenic implications of the model. HCV [E1.E2] molecule is modeled by using aa205–319 of E1 to aa421–716 of E2. Published experimental data were used to further refine the [E1.E2] model. Based on the model, we predict 77 exposed residues and several antigenic sites within the [E1.E2] that could serve as vaccine epitopes. This study identifies eight peptides which have antigenic propensity and have two or more sequentially exposed amino acids and 12 singular sites are under negative selection pressure that can serve as vaccine or therapeutic targets. Our special interest is ₂₈₅FLVGQLFTFSPRRHW₂₉₉ which has five negatively selected sites (L286, V287, G288, T292, and G303) with three of them sequential and four amino acids exposed (F285, L286, T292, and R296). This peptide in the E1 protein maps to dengue envelope vaccine target identified previously by our group. Our model provides for the first time an overall view of both the HCV envelope proteins thereby allowing researchers explore structure-based drug design approaches.     

细胞受体介导的疱疹病毒侵入机制

疱疹病毒(Herpesviruses)是一类较大的双链DNA囊膜病毒,广泛感染人和多种动物,以皮肤、粘膜和神经组织的疱疹性病变为特征。病毒侵入宿主细胞为整个复制周期的首要环节,而受体是介导病毒吸附、内化以及膜融合等侵入过程的关键宿主因子。因此,对受体的研究已经成为了解病毒侵入机制以及开发抗病毒制剂的突破口。本综述介绍了与疱疹病毒侵入相关的受体分子以及在这些分子介导下的跨膜机制,同时讨论了目前疱疹病毒侵入方面仍存在的问题和未来的研究方向。     

Subunit-selective role of the M3 transmembrane domain of the nicotinic acetylcholine receptor in channel gating

The nicotinic acetylcholine receptor (AChR) can be either hetero-pentameric, composed of α and non-α subunits, or homo-pentameric, composed of α7 subunits. To explore the subunit-selective contributions of transmembrane domains to channel gating we analyzed single-channel activity of chimeric muscle AChRs. We exchanged M3 between α1 and ? or α7 subunits. The replacement of M3 in α1 by ?M3 significantly alters activation properties. Channel activity appears as bursts of openings whose durations are 20-fold longer than those of wild-type AChRs. In contrast, 7-fold briefer openings are observed in AChRs containing the reverse ? chimeric subunit. The duration of the open state decreases with the increase in the number of α1M3 segments, indicating additive contributions of M3 of all subunits to channel closing. Each α1M3 segment decreases the energy barrier of the closing process by ∼ 0.8 kcal/mol. Partial chimeric subunits show that small stretches of the M3 segment contribute additively to the open duration. The replacement of α1 sequence by α7 in M3 leads to 3-fold briefer openings whereas in M1 it leads to 10-fold prolonged openings, revealing that the subunit-selective role is unique to each transmembrane segment.     

Expanding the Proteome of an RNA Virus by Phosphorylation of an Intrinsically Disordered Viral Protein

The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using ¹⁵N-, ¹³C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome.     

HIV进入抑制剂的研究进展

随着对HIV进入细胞过程的了解,各种进入抑制剂相继问世,目前主要有三大类:吸附抑制剂、辅助受体抑制剂和融合抑制剂.对其中具有代表性的进入抑制剂研究进展进行了介绍,一些进入抑制剂已经进入到了临床试验阶段,其中融合抑制剂T20在2003年便被FDA批准可同其他ARTs联合用于治疗HIV感染者,CCR5拮抗剂Maraviro...     

Increasing rate of cleavage at boundary between non-structural proteins 4B and 5A inhibits replication of hepatitis C virus

In hepatitis C virus, non-structural proteins are cleaved from the viral polyprotein by viral encoded proteases. Although proteolytic processing goes to completion, the rate of cleavage differs between different boundaries, primarily due to the sequence at these positions. However, it is not known whether slow cleavage is important for viral replication or a consequence of restrictions on sequences that can be tolerated at the cleaved ends of non-structural proteins. To address this question, mutations were introduced into the NS4B side of the NS4B5A boundary, and their effect on replication and polyprotein processing was examined in the context of a subgenomic replicon. Single mutations that modestly increased the rate of boundary processing were phenotypically silent, but a double mutation, which further increased the rate of boundary cleavage, was lethal. Rescue experiments relying on viral RNA polymerase-induced error failed to identify second site compensatory mutations. Use of a replicon library with codon degeneracy did allow identification of second site compensatory mutations, some of which fell exclusively within the NS5A side of the boundary. These mutations slowed boundary cleavage and only enhanced replication in the context of the original lethal NS4B double mutation. Overall, the data indicate that slow cleavage of the NS4B5A boundary is important and identify a previously unrecognized role for NS4B5A-containing precursors requiring them to exist for a minimum finite period of time.     

Structure–activity studies of (−)-epigallocatechin gallate derivatives as HCV entry inhibitors

Preventing viral entry into cells is a recognized approach for HIV therapy and has attracted attention for use against the hepatitis C virus (HCV). Recent reports described the activity of (−)-epigallocatechin gallate (EGCG) as an inhibitor of HCV entry with modest potency. EGCG is a polyphenolic natural product with a wide range of biological activity and unfavorable pharmaceutical properties. In an attempt to identify more drug-like EGCG derivatives with improved efficacy as HCV entry inhibitors, we initiated structure–activity investigations using semi-synthetic and synthetic EGCG analogs. The data show that there are multiple regions in the EGCG structure that contribute to activity. The gallate ester portion of the molecule appears to be of particular importance as a 3,4-difluoro analog of EGCG enhanced potency. This derivative and other active compounds were shown not to be cytotoxic in Huh-7 cell culture. These data suggest that more potent, non-cytotoxic EGCG analogs can be prepared in an attempt to identify more drug-like candidates to treat HCV infection by this mechanism.     

国内鱼类病毒病研究进展

病毒性疾病是危害水生动物最严重的一类疾病,是水产养殖业发展的重大障碍。近年来,中国水生动物病毒病的研究取得了一定成就,但是还有许多方面的研究有待加强,有很多未知领域需要进行探索与研究。本文综述了近年来国内鱼类病毒病的研究进展,主要介绍了草鱼出血病、传染性胰脏坏死病、淋巴囊肿病、传染性脾肾坏死病、鳗鲡病毒病、真鲷病毒病等不同鱼类病毒病的临床症状、流行病学、检测手段、致病机理、防治措施以及引起该病的病原体的形态结构、理化性质等,旨在为鱼类病毒病的防治提供参考。     

Understanding hepatitis C viral dynamics with direct-acting antiviral agents due to the interplay between intracellular replication and cellular infection dynamics

The current paradigm for modeling viral kinetics and resistance evolution after treatment initiation considers only the level of circulating virus and cellular infection (CI model), while the intra-cellular level is disregarded. This model was successfully used to explain HIV dynamics and Hepatitis C virus (HCV) dynamics during interferon-based therapy. However, in the new era of direct-acting antiviral agents (DAAs) against HCV, viral kinetics is characterized by a more rapid decline of the wild-type virus as well as an early emergence of resistant strains that jeopardize the treatment outcome. Although the CI model can be extended to describe these new kinetic patterns, this approach has qualitative and quantitative limitations. Instead, we suggest that a more appropriate approach would consider viral dynamics at the cell infection level, as done currently, as well as at the intracellular level. Indeed, whereas in HIV integrated DNA serves as a static replication unit and mutations occur only once per infected cell, HCV replication is deeply affected by DAAs and furthermore processes of resistance evolution can occur at the intra-cellular level with a faster time-scale.We propose a comprehensive model of HCV dynamics that considers both extracellular and intracellular levels of infection (ICCI model). Intracellular viral genomic units are used to form replication units, which in turn synthesize genomic units that are packaged and secreted as virions infecting more target cells. Resistance evolution is modeled intra-cellularly, by different genomic- and replication-unit strains with particular relative-fitness and drug sensitivity properties, allowing for a rapid resistance takeover.Using the ICCI model, we show that the rapid decline of wild-type virus results from the ability of DAAs to destabilize the intracellular replication. On the other hand, this ability also favors the rapid emergence, intracellularly, of resistant virus. By considering the interaction between intracellular and extracellular infection we show that resistant virus, able to maintain a high level of intracellular replication, may nevertheless be unable to maintain rapid enough de novo infection rate at the extracellular level. Hence this model predicts that in HCV, and contrary to our experience with HIV, the emergence of productively resistant virus may not systematically prevent from a viral decline in the long-term. Thus, the ICCI model can explain the transient viral rebounds observed with DAA treatment as well as the viral resistance found in most patients with viral relapse at the end of DAA combination therapy.     

Hepatitis C virus NS5A protein interacts with phosphatidylinositol 4-kinase type IIIalpha and regulates viral propagation

Hepatitis C Virus (HCV) nonstructural 5A (NS5A) is a pleiotropic protein involved in viral RNA replication and modulation of the cellular physiology in HCV-infected cells. To elucidate the mechanisms of the HCV life cycle, we identified cellular factors interacting with the NS5A protein in HCV-infected cells. Huh7.5 cells were electroporated with HCV Jc1 RNA. Cellular factors associated with HCV NS5A were identified by immunoprecipitation with Dynabead-conjugated NS5A antibody and LC-MS/MS. Phosphatidylinositol 4-kinase type IIIα (PI4KIIIα) was identified as a binding partner for the NS5A protein. NS5A derived from both genotypes 1b and 2a interacted with PI4KIIIα. NS5A interacted with PI4KIIIα through amino acids 401-600 of PI4KIIIα and domain I of NS5A. Interference of the protein interaction between NS5A and PI4KIIIα decreased HCV propagation. Knockdown of PI4KIIIα significantly reduced HCV replication in Huh7 cells harboring the subgenomic replicon and in Huh7.5 cells infected with cell culture grown virus (HCVcc). Silencing of PI4KIIIα further inhibited HCV release into the tissue culture medium. NS5A may recruit PI4KIIIα to the HCV RNA replication complex. These data suggest that PI4KIIIα is an essential host factor that supports HCV proliferation and therefore PI4KIIIα may be a legitimate target for anti-HCV therapy.     

C-type lectins do not act as functional receptors for filovirus entry into cells

Cellular C-type lectins have been reported to facilitate filovirus infection by binding to glycans on filovirus glycoprotein (GP). However, it is not clearly known whether interaction between C-type lectins and GP mediates all the steps of virus entry (i.e., attachment, internalization, and membrane fusion). In this study, we generated vesicular stomatitis viruses pseudotyped with mutant GPs that have impaired structures of the putative receptor binding regions and thus reduced ability to infect the monkey kidney cells that are routinely used for virus propagation. We found that infectivities of viruses with the mutant GPs dropped in C-type lectin-expressing cells, parallel with those in the monkey kidney cells, whereas binding activities of these GPs to the C-type lectins were not correlated with the reduced infectivities. These results suggest that C-type lectin-mediated entry of filoviruses requires other cellular molecule(s) that may be involved in virion internalization or membrane fusion.     

The Measles Virus Nucleocapsid Protein Tail Domain Is Dispensable for Viral Polymerase Recruitment and Activity

Paramyxovirus genomes are ribonucleoprotein (RNP) complexes consisting of nucleoprotein (N)-encapsidated viral RNA. Measles virus (MeV) N features an amino-terminal RNA-binding core and a 125-residue tail domain, of which only the last 75 residues are considered fully mobile on the nucleocapsid surface. A molecular recognition element (MoRE) domain mediates binding of the viral phosphoprotein (P). This P N-tail interaction is considered instrumental for recruiting the polymerase complex to the template. We have engineered MeV N variants with tail truncations progressively eliminating the MoRE domain and upstream tail sections. Confirming previous reports, RNPs with N truncations lacking the carboxyl-terminal 43-residues harboring the MoRE domain cannot serve as polymerase template. Remarkably, further removal of all tail residues predicted to be surface-exposed significantly restores RNP bioactivity. Insertion of structurally dominant tags into the central N-tail section reduces bioactivity, but the negative regulatory effect of exposed N-tail stems is sequence-independent. Bioactive nucleocapsids lacking exposed N-tail sections are unable to sustain virus replication, because of weakened interaction of the advancing polymerase complex with the template. Deletion of the N-MoRE-binding domain in P abrogates polymerase recruitment to standard nucleocapsids, but polymerase activity is partially restored when N-tail truncated RNPs serve as template. Revising central elements of the current replication model, these data reveal that MeV polymerase is capable of productively docking directly to the nucleocapsid core. Dispensable for polymerase recruitment, N-MoRE binding to P-tail stabilizes the advancing polymerase-RNP complex and may rearrange unstructured central tail sections to facilitate polymerase access to the template.     

SR-BI及其伴侣分子PDZK1在HCV入侵中的作用

B族Ⅰ型清道夫受体（scavenger receptor class B type I,SR-BI）是丙型肝炎病毒（hepatitis C virus,HCV）的受体之一,可以与HCV的包膜蛋白E2结合,介导病毒颗粒进入宿主细胞。伴侣分子PDZK1（PDZdomain containing 1）是一个含有4个PDZ结构域的支架蛋白,其第一个PDZ结构域可以与SR-BI的C端结合,调节其稳定表达和正确定位。研究发现PDZK1基因敲除以后,HCVcc（cell culture produced HCVvirus）和HCVpp（HCV pseudotype particles）的感染性明显下降;重新转入PDZK1后,可以部分恢复感染性。研究表明PDZK1可促进HCV入侵并可能是通过与SR-BI的相互作用介导的。伴侣分子对受体分子的调节在HCV入侵中的作用可能成为HCV治疗的潜在靶标,有助于开发新的治疗方法。     

我国水生动物病毒病研究概况

80年代以来,我国水产业迅速发展,并且在农业产值中所占的比重逐年上升,已成为我国大农业的四大支柱产业(粮食、肉类、水产和禽蛋)之一。1998年,我国水产品年产量达3800万t,但令人遗憾的是水生动物疾病,尤其是因病毒引起的爆发性流行病明显增多,危害极为严重。水生动物分类地位相差甚远,除了鱼之外,还包括虾、贝等无脊椎动物和蛙、鳖等低等脊椎动物。自从Wolf分离到第一株鱼类病毒以来[1],迄今见报道的鱼病毒已超过70种。     

Movement of DNA across the chloroplast envelope: Implications for the transfer of promiscuous DNA

Little is known about the mechanistic basis for the movement of promiscuous nucleic acids across cell membranes. To address this problem we sought conditions that would permit the entry of plasmid DNA into isolated, intact pea chloroplasts. DNA uptake did not occur normally, but was induced by hypotonic treatments, by incubation with millimolar levels of Mg²⁺, or by heat shock at 42 °C. These results are consistent with DNA movement being permitted by conditions that transiently alter the permeability of the chloroplast envelope. Plant cells are subject to osmotic tensions and/or conditions inducing polymorphic changes in the membranes, such as those used in the present study, under several environmental stresses. In an evolutionary time frame, these phenomena may provide a mechanism for the transfer of promiscuous nucleic acids between organelles.Abbreviations PEG polyethylene glycol - T-DNA transferred DNATo Dan Arnon; a superb scientist whose findings opened up enormous fields of research in photosynthesis, and a good friend.