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.     

Virus factories: associations of cell organelles for viral replication and morphogenesis

Genome replication and assembly of viruses often takes place in specific intracellular compartments where viral components concentrate, thereby increasing the efficiency of the processes. For a number of viruses the formation of 'factories' has been described, which consist of perinuclear or cytoplasmic foci that mostly exclude host proteins and organelles but recruit specific cell organelles, building a unique structure. The formation of the viral factory involves a number of complex interactions and signalling events between viral and cell factors. Mitochondria, cytoplasmic membranes and cytoskeletal components frequently participate in the formation of viral factories, supplying basic and common needs for key steps in the viral replication cycle.     

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.     

Hydrophobic segment of dengue virus C protein. Interaction with model membranes

Abstract

Dengue virus (DENV) C protein is essential for viral assembly. DENV C protein associates with intracellular membranes through a conserved hydrophobic domain and accumulates around endoplasmic reticulum-derived lipid droplets which could provide a platform for capsid formation during assembly. In a previous work we described a region in DENV C protein which induced a nearly complete membrane rupture of several membrane model systems, which was coincident with the theoretically predicted highly hydrophobic region of the protein. In this work we have carried out a study of the binding to and interaction with model biomembranes of a peptide corresponding to this DENV C region, DENV2_C6. We show that DENV2_C6 partitions into phospholipid membranes, is capable of rupturing membranes even at very low peptide-to-lipid ratios and its membrane-activity is modulated by lipid composition. These results identify an important region in the DENV C protein which might be directly implicated in the DENV life cycle through the modulation of membrane structure.     

Pathology of hepatitis C

Abstract: The basic morphological patterns of acute or chronic viral hepatitides are very similar, irrespective of the causative hepatitis viruses A, B, C, D or E. In addition, however, acute and chronic hepatitis C shows characteristic, although not pathognomonic histological changes. These consist of lymphoid aggregates in portal tracts, sometimes with germinal centers, damage of bile duct epithelium, and micro- or macrovesicular steatosis of hepatocytes. A combination of two of these three characteristic alterations is seen in over half of the patients with chronic hepatitis C and is helpful in the histological diagnosis of the disease.     

间接核酸杂交方法的建立

建立了一种新的核酸杂交手段一间接核酸杂交方法,其突出的优点是用一种共同的核酸标记物就可检查不同的基因组或不同的基因。我们重组乙型肝炎病毒或EB病毒的核酸片段于噬菌体M_(13)mp8载体,以此重组的单链DNA为第一夹心层,用~(32)P标记的双链噬菌体DNA作为共同探针,检查乙型肝炎病毒和EB病毒的核酸,获得满意的结果。应用该法进行细胞内的原位杂交,检查细胞内存在的EB病毒基因效果亦佳。     

Zika Virus Dependence on Host Hsp70 Provides a Protective Strategy against Infection and Disease

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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.     

A novel approach to achieving modular retrovirus clearance for a parvovirus filter

Viral filtration is routinely incorporated into the downstream purification processes for the production of biologics produced in mammalian cell cultures (MCC) to remove potential viral contaminants. In recent years, the use of retentive filters designed for retaining parvovirus (～20 nm) has become an industry standard in a conscious effort to further improve product safety. Since retentive filters remove viruses primarily by the size exclusion mechanism, it is expected that filters designed for parvovirus removal can effectively clear larger viruses such as retroviruses (～100 nm). In an attempt to reduce the number of viral clearance studies, we have taken a novel approach to demonstrate the feasibility of claiming modular retrovirus clearance for Asahi Planova 20N filters. Porcine parvovirus (PPV) and xenotropic murine leukemia virus (XMuLV) were co‐spiked into six different feedstreams and then subjected to laboratory scale Planova 20N filtration. Our results indicate that Planova 20N filters consistently retain retroviruses and no retrovirus has ever been detected in the filtrates even when significant PPV breakthrough is observed. Based on the data from multiple in‐house viral validation studies and the results from the co‐spiking experiments, we have successfully claimed a modular retrovirus clearance of greater than 6 log₁₀ reduction factors (LRF) to support clinical trial applications in both USA and Europe. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:79–85, 2014     

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.     

Infection of macaques with a molecular clone,SHIVSF33A2, provides evidence for tissue specific variants

Infection of rhesus macaques with chimeric simian-human immunodeficiency viruses (SHIV) is an established model to study acquired immunodeficiency syndrome (AIDS) pathogenesis. Such a controlled system allows for detailed analysis of the molecular determinants of viral pathogenesis in addition to studying host-specific immune responses that modulate disease progression. Furthermore, the use of a pathogenic molecular clone affords the opportunity to study both viral evolution within a host and to examine the generation of tissue specific variants. In this report we describe viral diversification within tissues of two rhesus macaques infected intravenously with the CXCR4-specific molecular clone SHIVSF33A2. Heteroduplex tracking analysis (HTA) was used to determine the complexity of viral DNA within distinct lymphoid tissues. Not surprising, heterogeneity of the proviral quasispecies in tissues obtained during the acute infection was limited. However, tissues obtained at necropsy harbored a more diverse and often different population of env variants. As the inoculating virus is a molecular clone, the variants generated are likely due to the presence of tissue specific selective forces rather than a founder's effect.     

The Viral Oncoprotein HBx of Hepatitis B virus Promotes the Growth of Hepatocellular Carcinoma through Cooperating with the Cellular Oncoprotein RMP

The smallest gene HBx of Hepatitis B virus (HBV) is recognized as an important viral oncogene (V-oncogene) in the hepatocarcinogenesis. Our previous work demonstrated that RMP is a cellular oncogene (C-oncogene) required for the proliferation of hepatocellular carcinoma (HCC) cells. Here we presented the collaboration between V-oncogene HBx and C-oncogene RMP in the development of HCC. The coexpression of HBx and RMP resulted in the cooperative effect of antiapoptosis and proliferation of HCC cells. In vivo, overexpression of RMP accelerated the growth of HBx-induced xenograft tumors in nude mice and vice versa HBx promoted the growth of RMP-driven xenograft tumors. Although HBx didn''t regulate the expression of RMP, HBx and RMP interact with each other and collocalized in the cytoplasm of HCC cells. HBx and RMP collaboratively inhibited the expression of apoptotic factors and promoted the expression of antiapoptotic factors. This finding suggests that HBV may induce, or at least partially contributes to the carcinogenesis of HCC, through its V-oncoprotein HBx interacting with the C-oncoprotein RMP.     

Validation of biopharmaceutical purification processes for virus clearance evaluation

Any biopharmaceutical product that has involved the use of animal-derived material during the manufacturing process has the potential to be contaminated with animal viruses. To ensure safety of these products, extensive testing is performed on the starting materials, such as the cell banks, and on the raw materials used in manufacture. Additional testing is also performed at various stages of production and, in some cases, on the final product as well. Because of inherent limitations in direct testing methods, the capacity of the downstream purification process to remove/inactivate potential viral contaminants is also studied to give an extra degree of assurance that the final product will be free of infectious viruses.     

基因治疗中关于非病毒载体的最新设计策略

基因转移的非病毒技术近来发展迅速。与病毒转染细胞的方法相比,非病毒转移方法比较简便,安全,毒性小。这种方法大体可分为两种：完全非病毒方法和病毒增强的转移方式。本文主要介绍一些最近两三年来新兴的技术方案的优点和不足。     

Viral detection by electron microscopy: past, present and future

Viruses are very small and most of them can be seen only by TEM (transmission electron microscopy). TEM has therefore made a major contribution to virology, including the discovery of many viruses, the diagnosis of various viral infections and fundamental investigations of virus-host cell interactions. However, TEM has gradually been replaced by more sensitive methods, such as the PCR. In research, new imaging techniques for fluorescence light microscopy have supplanted TEM, making it possible to study live cells and dynamic interactions between viruses and the cellular machinery. Nevertheless, TEM remains essential for certain aspects of virology. It is very useful for the initial identification of unknown viral agents in particular outbreaks, and is recommended by regulatory agencies for investigation of the viral safety of biological products and/or the cells used to produce them. In research, only TEM has a resolution sufficiently high for discrimination between aggregated viral proteins and structured viral particles. Recent examples of different viral assembly models illustrate the value of TEM for improving our understanding of virus-cell interactions.     

Using viral genomics to develop viral gene products as a novel class of drugs to treat human ailments

A novel strategy for drug discovery and advancement has been developed by exploiting viral genomics. By manipulating cell function through the therapeutic administration of specific gene products from viruses, an important new class of therapeutics could be developed for the treatments of major human diseases. As it may turn out in the human genome arena, the groups which can establish a stronghold in the field would be in the best position to exploit this new drug development strategy.