Evolution of viral structure

Viruses vastly outnumber their host cells and must present a huge selective pressure. It is also becoming evident that only a small percent of the eukaryotic genome codes for molecules involved in cellular structures and functions, and that much of the remainder may have a viral origin. Viruses clearly play a central role in the biosphere, but how is this viral world organized? Classification was originally based on virus morphology and the particular host infected, but now there is an increasing trend to rely on sequence information. The type of genome (e.g., RNA or DNA, single- or double-stranded) provides fundamental classification criteria, while sequence comparisons can provide fine mapping for closely related viruses. However, it is currently very difficult to identify long-range evolutionary relationships. We present here a different approach, based on the idea that each virus has an innate "self." When the structures and functions characteristic of this "self" are identified, then they uncover relationships beyond those accessible from sequence information alone. The new approach is illustrated by sketching some possible viral lineages. We propose that urviruses were present before the division of cellular life into its current domains, and that the viral world has lineages that can be traced back to the root of the universal tree of life.     

Synthetic protocells interact with viral nanomachinery and inactivate pathogenic human virus

We present a new antiviral strategy and research tool that could be applied to a wide range of enveloped viruses that infect human beings via membrane fusion. We test this strategy on two emerging zoonotic henipaviruses that cause fatal encephalitis in humans, Nipah (NiV) and Hendra (HeV) viruses. In the new approach, artificial cell-like particles (protocells) presenting membrane receptors in a biomimetic manner were developed and found to attract and inactivate henipavirus envelope glycoprotein pseudovirus particles, preventing infection. The protocells do not accumulate virus during the inactivation process. The use of protocells that interact with, but do not accumulate, viruses may provide significant advantages over current antiviral drugs, and this general approach may have wide potential for antiviral development.     

From deep sequencing to viral tagging: Recent advances in viral metagenomics

Culture‐independent high‐throughput sequencing has provided unprecedented insights into microbial ecology, particularly for Earth's most ubiquitous and diverse inhabitants – the viruses. A plethora of methods now exist for amplifying the vanishingly small amounts of nucleic acids in natural viral communities in order to sequence them, and sequencing depth is now so great that viral genomes can be detected and assembled even amid large concentrations of non‐viral DNA. Complementing these advances in amplification and sequencing is the ability to physically link fluorescently labeled viruses to their host cells via high‐throughput flow sorting. Sequencing of such isolated virus–host pairs facilitates cultivation‐independent exploration of the natural host range of viruses. Within the next decade, as these technologies become widespread, we can expect to see a systematic expansion of our knowledge of viruses and their hosts.     

Modern genetic approaches to searching for targets for medicinal preparations

In spite of a vast number of drug preparations used in medicine, advances in treating most socially important human diseases remain modest. Historically, many drugs were developed without clear understanding of the mechanisms of their action and were intended only for correcting symptoms of the disease. Identification of molecular targets in pharmacological screening new pharmaceuticals plays a key role not only in defining the strategy of the treatment, but also in understanding the general development of the disease. Sequencing of the genomes of various organisms, human in particular, and the development of new modern techniques of research have created the prerequisites for targeted screening for genes that are potentially interesting for development of new drugs.     

Pharmacophore modeling,resistant mutant isolation,docking, and MM-PBSA analysis: Combined experimental/computer-assisted approaches to identify new inhibitors of the bovine viral diarrhea virus (BVDV)

Starting from a series of our new 2-phenylbenzimidazole derivatives, shown to be selectively and potently active against the bovine viral diarrhea virus (BVDV), we developed a hierarchical combined experimental/molecular modeling strategy to explore the drug leads for the BVDV RNA-dependent RNA-polymerase. Accordingly, a successful 3D pharmacophore model was developed, characterized by distinct chemical features that may be responsible for the activity of the inhibitors. BVDV mutants resistant to lead compounds in our series were then isolated, and the mutant residues on the viral molecular target, the RNA-dependent RNA-polymerase, were identified. Docking procedures upon pharmacophoric constraints and mutational data were carried out, and the binding affinity of all active compounds for the RdRp were estimated. Given the excellent agreement between in silico and in vitro data, this procedure is currently being employed in the design a new series of more selective and potent BVDV inhibitors.     

衣壳蛋白靶向灭活 一种新型抗病毒策略

衣壳蛋白靶向灭活（CTVI）是一种新型抗病毒策略,它是通过将病毒衣壳蛋白与核酸酶（金黄色葡萄球菌和酸梅、核糖核酸酶Barnase、大肠杆菌RNase HI等）的融合蛋白装配到病毒粒子中,使核酸酶接触并降解病毒核酸,从而达到抑制病毒复制的目的。该策略已经在人免疫缺陷病毒、鼠白血病病毒、乙肝病毒、登革病毒等病毒的抗病毒研究中取得良好效果,展示了广阔的应用前景。     

Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2

Human angiotensin-converting enzyme 2 (ACE2) is a functional receptor for SARS coronavirus (SARS-CoV). Here we identify the SARS-CoV spike (S)-protein-binding site on ACE2. We also compare S proteins of SARS-CoV isolated during the 2002-2003 SARS outbreak and during the much less severe 2003-2004 outbreak, and from palm civets, a possible source of SARS-CoV found in humans. All three S proteins bound to and utilized palm-civet ACE2 efficiently, but the latter two S proteins utilized human ACE2 markedly less efficiently than did the S protein obtained during the earlier human outbreak. The lower affinity of these S proteins could be complemented by altering specific residues within the S-protein-binding site of human ACE2 to those of civet ACE2, or by altering S-protein residues 479 and 487 to residues conserved during the 2002-2003 outbreak. Collectively, these data describe molecular interactions important to the adaptation of SARS-CoV to human cells, and provide insight into the severity of the 2002-2003 SARS epidemic.     

Caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis

Viral infection constitutes an unwanted intrusion that needs to be eradicated by host cells. On one hand, one of the first protective barriers set up to prevent viral replication, spread or persistence involves the induction of apoptotic cell death that aims to limit the availability of the cellular components for viral amplification. On the other hand, while they completely depend on the host molecular machinery, viruses also need to evade the cellular responses that are meant to destroy them. The existence of numerous antiapoptotic products within the viral kingdom proves that apoptosis constitutes a major threat that should better be bypassed. Among the different strategies developed to deal with apoptosis, one is based on what viruses do best: backfiring the cell on itself. Several unrelated viruses have been described to take advantage of apoptosis induction by expressing proteins targeted by caspases, the key effectors of apoptotic cell death. Caspase cleavage of these proteins results in various consequences, from logical apoptosis inhibition to more surprising enhancement or attenuation of viral replication. The present review aims at discussing the characterization and relevance of this post-translational modification that adds a new complexity in the already intricate host–apoptosis–virus triangle.     

The role of viral mutation in the pathogenesis of chronic viral hepatitis

The quasispecies nature of hepatitis B and C virus (HBV, HCV) plays an important role in the pathogenesis, immune escape and drug resistance during chronic infection. Although there is still a lack of effective treatment for hepatitis C, a series of nucleoside analogs (NA) have been developed for the treatment of hepatitis B. NA resistant HBV mutants can accumulate during prolonged therapy and lead to the failure of anti-HBV therapy. Switching to other sensitive NAs can inhibit the emerged resistant mutants. Therefore, understanding the evolution of viral quasispecies under drug pressure is crucial for the establishment of antiviral strategy and the monitoring of antiviral process. Immune response and escape are complicated process, during which both host and virus factors may play their roles. Further understanding of the interaction and interrelationship between host and these viruses may lead to optimized prevention, diagnosis and treatment for chronic hepatitis.     

Relationships between proteasomes and viral gene products

The interrelationships between proteasomes and viral gene products are very complex. 20S proteasomes associate with a number of viral mRNAs which are cleaved by proteasome's associated endonuclease activity. In addition proteasome's endopeptidase activities are involved in the presentation of viral antigens. Viral proteins of different origin associate with the 20S and 26S complexes and interfere with their enzymatic activities. A major part of this review deals with the interactions between 20S proteasomes and the gene products of the human immunodeficiency virus (HIV) which has been studied in detail by our group.     

基因工程疫苗的病毒载体

病毒基因工程疫苗是以活病毒为载体将一段外源基因导入机体细胞内,并使外源基因维持较高水平的表达。通过使用复制型或复制缺陷型载体能使表达的抗原诱生机体产生相应的体液抗体,并能引起机体产生细胞介导的免疫反应及粘膜免疫反应。本文主要介绍有可能用于基因工程疫苗的DNA及RNA病毒载体构建及其应用。     

Expression of viral capsid protein antigen against Epstein-Barr virus in plastids of Nicotiana tabacum cv. SR1

Epstein-Barr virus (EBV) infects nearly 90% of adults worldwide and is the pathogenic source of a broad spectrum of malignancies originating from lymphoid and epithelial cells. Currently, no vaccine has been developed to immunologically inactivate this virus. In infected patients, anti-EBV viral capsid antigen (VCA) immunoglobins represent some of the useful diagnostic markers for carcinoma development. To demonstrate that the EBV VCA antigen can be produced in plants, the plastid genome of tobacco (Nicotiana tabacum cv. SR1) was transformed with a VCA-expressing cassette. The EBV VCA mRNA was actively transcribed in the transplastomic plants and antigen production was detected. This study indicates that plastid transformation could be a promising strategy in EBV VCA antigen production.     

Targeting host cofactors to inhibit viral infection

The majority of FDA-approved drugs indicated for the treatment of viral infections are inhibitors of viral proteins, of which the emergence of resistant strains is a major concern. This issue is exacerbated as most developed antiviral therapies are indicated for the treatment of viruses with error-prone replication. These problems may be addressed by the development of drugs that modulate the function of host factors involved in various aspects of a viral life cycle. Targeting host factors uncouples the mutation of a druggable protein gene from the replication and survival selection pressure exerted on a virus. Currently, a host-targeting antiviral (HTA), maraviroc, is approved for the treatment of human immunodeficiency virus (HIV) infection. In addition, several HTAs indicated for the treatment of hepatitis C virus (HCV) or HIV infection are at various stages of clinical evaluation. Targeting host factors is an attractive complement to therapies directly targeting a viral protein because of the expected higher genetic barrier for resistance and an overall increase in the diversity of treatment options. We examine how the integrated roles of emerging host cofactor screening approaches and drug development strategies may advance current treatment options.     

人类口服病毒疫苗的研究进展

研制能同时诱导有效黏膜免疫和系统免疫的疫苗是预防黏膜感染病原体的理想目标。消化道具有许多产生黏膜免疫的位点,包括口腔、胃和小肠等。理想的口服病毒疫苗不仅能诱导较好的局部及远端黏膜免疫,也能产生较好的系统免疫,而且还因为具有无痛接种、可自行服用等优势而备受关注。由于人消化道环境及黏膜免疫的复杂性,目前成功上市的人口服病毒疫苗仅限于3种减毒活疫苗。本文将从消化道黏膜免疫的特点、当前口服病毒疫苗种类及研究现状、口服病毒疫苗所面临的挑战等方面进行综述,期望对我国人口服病毒疫苗的研究和开发提供参考和借鉴。     

A database of recombinant viruses and recombinant viral vectors available from the RIKEN DNA bank

BACKGROUND: Viral vectors are required as gene-delivery systems for gene therapy and basic research. Recombinant adenoviruses (rAds) expressing genes of interest are being developed as research tools and many studies in vitro and in vivo have already been performed with such rAds. METHODS: Shuttle vectors for rAds were constructed with full-length cDNAs and rAds were generated in HEK293 cells by the COS-TPC method. The rAds and shuttle vectors were developed by the Japanese research community and deposited in the RIKEN DNA Bank (RDB; http://www.brc.riken.jp/lab/dna/en/) for distribution to the scientific community. The Recombinant Virus Database (RVD; http://www.brc.riken.jp/lab/dna/rvd/) was established at the RIKEN BioResource Center (BRC) in Japan as the source of information about and distribution of the various resources. RESULTS: The RIKEN BRC is releasing more than 300 recombinant viruses (RVs) and 500 shuttle vectors, as well as all related information, which is included in a newly established database, the RVD. The RVD consists of (i) information about the RVs, the inserted cDNAs and the shuttle vectors; (ii) data about sequence-tagged sites (STSs) that are markers of viral DNAs; and (iii) experimental protocols for the use of RVs. CONCLUSIONS: The new database and available resources should be very useful to scientists who are studying human gene therapy and performing related basic research. It is a web-interfaced flat-file database that can be accessed through the internet. Moreover, all of the resources deposited in the RDB, which is a public facility in Japan, are available to researchers around the world.     

Synergistic experimental/computational studies on arylazoenamine derivatives that target the bovine viral diarrhea virus RNA-dependent RNA polymerase

Starting from a series of arylazoenamine derivatives, shown to be selectively and potently active against the bovine viral diarrhea virus (BVDV), we developed a hierarchical combined experimental/molecular modeling strategy to explore the drug leads for the BVDV RNA-dependent RNA polymerase. Accordingly, BVDV mutants resistant to lead compounds in our series were isolated, and the mutant residues on the viral molecular target, the RNA-dependent RNA polymerase, were identified. Docking procedures upon previously identified pharmacophoric constraints and actual mutational data were carried out, and the binding affinity of all active compounds for the RdRp was estimated. Given the excellent agreement between in silico and in vitro data, this procedure is currently being employed in the design a new series of more selective and potent BVDV inhibitors.     

Molecular mechanisms for viral mimicry of a human cytokine: activation of gp130 by HHV-8 interleukin-6

Kaposi's sarcoma-associated herpesvirus (KSHV, or HHV-8) encodes a pathogenic viral homologue of human interleukin-6 (IL-6). In contrast to human IL-6 (hIL-6), viral IL-6 (vIL-6) binds directly to, and activates, the shared human cytokine signaling receptor gp130 without the requirement for pre-complexation to a specific alpha-receptor. Here, we dissect the biochemical and functional basis of vIL-6 mimicry of hIL-6. We find that, in addition to the "alpha-receptor-independent" tetrameric vIL-6/gp130 complex, the viral cytokine can engage the human alpha-receptor (IL-6Ralpha) to form a hexameric vIL-6/IL-6Ralpha/gp130 complex with enhanced signaling potency. In contrast to the assembly sequence of the hIL-6 hexamer, the preformed vIL-6/gp130 tetramer can be decorated with IL-6Ralpha, post facto, in a "vIL-6-dependent" fashion. A detailed comparison of the viral and human cytokine/gp130 interfaces indicates that vIL-6 has evolved a unique molecular strategy to interact with gp130, as revealed by an almost entirely divergent structural makeup of its receptor binding sites. Viral IL-6 appears to utilize an elegant combination of both convergent, and unexpectedly divergent, molecular strategies to oligomerize gp130 and activate similar downstream signaling cascades as its human counterpart.     

Host selenium status selectively influences susceptibility to experimental viral myocarditis

The purpose of the present work was to determine whether dietary selenium (Se) deficiency could influence the injurious effect of human viruses other than Coxsackie virus B3 (CVB3) on mouse heart. Weanling C3H/HeN mice were fed a Se-deficient or Se-adequate diet for 4 wk and then were inoculated intraperitoneally with one of the following viruses: Coxsackie virus B1 (CVB1), echovirus 9 (EV9), Coxsackie virus A9 (CVA9), or herpes simplex 1 (HSV1). Polio virus 1 (PV1) was employed as a negative control. Prior to inoculation, mean serum Se levels were 430 versus 61 ng/mL in adequate versus deficient mice, respectively. Ten days later, hearts were removed and processed by routine histological procedures. Cardiac lesions were scored according to the number and size of myocarditic foci. Significantly greater heart damage resulting from CVB1 and EV9 was observed in Se-deficient than in Se-adequate mice, and the Se status had no influence on CVA9-induced myocarditis. In contrast, heart damage caused by HSV1 was significantly milder in Se-deficient than in Se-adequate mice. Therefore, it may be concluded that the Se status of the murine host selectively influences the degree of viral-induced myocarditic lesions.