Rhythm of a life within life: role of viral suppressors in hijacking the host cell

Virus–host interaction at cellular and intra-cellular level is a constantly evolving process which results either in the host to resist or the virus to break host immunity and to establish the disease. We have put extensive efforts to understand the genomic organization and gene functions of important viral proteins involved in resisting or avoiding host antiviral responses mediated by RNA interference or Ubiquitin–Proteasome Pathway. Nearly two decades of dedicated research on three agriculturally important viruses revealed genetic and epigenetic regulation of host to induce the defense/immunity responses. The microRNA and auxin regulated development of disease symptoms and the role of temperature in optimizing the interactions of viral protein with host small RNAs are intriguing observations. Owing to the complexity of the dynamic interactions between plant and virus, this research field will always be challenging and fascinating.

     

Possible role of SV40 T antigen in cell transformation]

The effect of purified SV40 T antigen on DNA synthesis in isolated nuclei from the confluent culture of CV-1 cells was studied. In the presence of T antigen the incorporation of [3H]TTP into DNA was found to be 2 to 3 times as high as in the control nuclei. The resulting labelled DNA was subjected to alkaline sucrose gradient centrifugation, which revealed the presence of 4S DNA species, corresponding to Okazaki fragments of animal cells. The latter finding suggests a replicative mode of DNA synthesis induced by T antigen. T antigen isolated from the cells infected with SV40 tsA-mutant and kept at a nonpermissive (41 degrees) temperature fails to stimulate DNA synthesis in isolated nuclei from resting cells. On storage at 4 degrees SV40 T antigen gradually loses its ability to stimulate DNA synthesis and by the 8th day even suppresses it when tested on isolated nuclei from a growing cell culture. No effect of T antigen on the endonuclease-induced reparative synthesis of DNA could be observed. The data described suggest that T antigen is directly involved in the control of DNA synthesis in the cells infected or transformed with SV40.     

Adenovirus 2 Ip+ locus codes for a 19 kd tumor antigen that plays an essential role in cell transformation

Adenovirus 2 large plaque (Ip) mutants produce large clear plaques on human KB cells. These mutants are shown to be defective in inducing transformation of the established rat embryo cell line 3Y1. The Ip mutation was localized within one of the two transforming early gene blocks, E1b (map position 4.5 to 11.2) which codes for two major T antigens of 53 kd and 19 kd by marker transfer. The mutational defects in mutants Ip3 and Ip5 were analyzed by DNA sequence analysis and by analysis of viral E1 proteins. These results reveal that Ip3 and Ip5 mutations map within the 19 kd tumor antigen coding region. Mutant Ip3 has a single base pair change at the N terminus of 19 kd polypeptide, resulting in the substitution of valine for alanine. Mutant Ip5 has two mutational changes, one of which results in the substitution of tyrosine for aspartic acid near the N-terminal region. The second mutation changes the termination codon into a leucine codon, increasing the size of the 19 kd tumor antigen. These results provide direct genetic evidence for an essential role of the 19 kd tumor antigen in cell transformation and indicate that the N-terminal region of the 19 kd tumor antigen is an essential function domain for the induction of cell transformation.     

Cell cycle transformation in cell differentiation

A question was posed as to how the multicomponent and polyfunctional organelle dynamically changes during metazoan ontogenesis. The centrosome structure is gradually formed and its functions are switched on during early embryogenesis, one of which is the cell center formation. During cell differentiation, the condition of the cell center and surrounding structures may be different: first, the cell center is quite distinct; second, the cell center is absent due to redistribution of the centers of microtubule organization; third, the cell center disappears due to reversible or irreversible inactivation of the centrosome and other centers of microtubule organization. The assembly of the common Golgi complex is not directed directly to the cell center presence. In some cell types, the Golgi complex is topologically associated with the cell center, while in others it is represented by individual dictyosomes despite the cell center presence. In some other cell types, the common Golgi complex is assembled without the cell center, but in the presence of microtubules that are formed by noncentrosome centers of microtubule organization. In still others, degradation of both the cell center and the common Golgi complex takes place in the case of centrosome inactivation.     

植物生活史型的划分及其相互转化

基于环境条件的变化,分析了植物在完成生活史过程中,营养生长、克隆生殖和有性生殖过程的权衡的结果直接导致的植物生活史型形成以及相互转化过程。根据胁迫和扰动程度将植物的生境划分为基本生境型、特殊过渡型和过渡型,其中过渡型(EDF)在自然界中具有普遍意义;与生境过渡型(EDF)相对应,植物形成的生活史型也为过渡生活史型(VCS),其基本型V、C和S以及特殊过渡型VC、VS和CS是其特例。植物的生活史型过渡型(VCS)具有普适性意义,在生境调控下的任一种植物生活史型均可表示为Vx1Cx2Sx3。植物生活史型的相互转化与其生境类型的相互转变是完全对应的,这种相互转化的过程和途径是复杂的,且具有可逆性的特点。     

The role of cell contact in the life cycle of some dinoflagellate species

It is suggested, based on published and experimental data, thatinter- and intra-specific cell contact plays an important rolein the life cycle of some dinoflagellate species.     

Spike protein, S, of human coronavirus HKU1: role in viral life cycle and application in antibody detection

We recently described the discovery, genome, clinical features, genotypes and evolution of a novel and global human respiratory virus named human coronavirus HKU1 (HCoV-HKU1) which is not yet culturable. We expressed a C-terminal FLAG-tagged CoV-HKU1 spike (S) protein by the Semliki Forest Virus (SFV) system and investigated its maturation profile. Pulse chase labeling revealed that S-FLAG was expressed as high-mannose N-glycans of monomers and trimers. It was predominantly cleaved into subdomains S1 and S2 during maturation. S1 was secreted into the medium. Immunofluorescence analysis visualized S along the secretory pathway from endoplasmic reticulum to plasma membrane. Cleavage of S and release of HCoV-HKU1 S pseudotyped virus were inhibited by furin or furin-like enzyme inhibitors. The cell-based expressed full-length S-FLAG could be recognized by the convalescent serum obtained from a patient with HCoV-HKU1 pneumonia. The data suggest that the native form of HCoV-HKU1 spike expressed in our system can be used in developing serological diagnostic assay and in understanding the role of S in the viral life cycle.     

A consideration of the role of cell surface macromolecules in the process of viral transformation

Summary There is extensive physiological evidence implicating the cell surface as the key organelle which mediates the cell:cell interactions which underlie both normal and neoplastic growth. This information has now been supplemented with biochemical and biophysical data which indicates that surface macromolecules, in particular the heteroglycans of transformed cells, differ from those which lie at the periphery of normal cells. In the case of cells neoplastically transformed by most tumour viruses it is clear that the small virus genome (2–5×10⁶ daltons) cannot carry the total genetic information to accomodate these various biochemical modifications, if indeed they are encoded in separate genes (1). To examine the part played in transformation by cellular genes coding for surface heteroglycan formation, we have turned to a study of SV-3T3 cells (ts H6-15) which are temperature-sensitive for expression of the transformed cell phenotype (2). The data show that cells grown under conditions permissive and non-permissive for such expression exhibit the same pattern of formation of glycolipids, and of the majority of the polypeptides of the plasma membrane. There are, however, significant differences in the synthesis of some glycopeptides. A large molecular weight, trypsin-labile glycopeptide, present at the surface of untransformed fibroblasts but barely measurable in some of their virus-transformed derivatives (3), was detected, essentially at the same level, at the surface ofts H6-15 cells grown at the permissive and non-permissive temperatures. The significance of these observations is discussed. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Calpain: a role in cell transformation and migration

Calpains represent a well conserved family of calcium-dependent proteolytic enzymes. Recent progress in determining the three-dimensional crystal structure of calpains and generation of calpain knock out animals have significantly advanced our understanding of both the activation mechanism and physiological role of this protease family. Studies applying molecular intervention strategies and genetic ablation of calpain now provide indisputable evidence that calpain activity contributes to remodelling of the actin cytoskeleton, cell migration and oncogenic transformation. Src and epidermal growth factor receptor (EGFR) stimulated cell motility is dependent upon calpain activation. In addition, calpain promotes accelerated cell-cycle progression and anchorage-independent growth of Src transformed cells. In vivo studies demonstrate a link between calpain expression levels and activity with tumour development and invasion. Thus, recent investigations suggest that the role of calpain in promoting cell transformation and cell migration may have important in vivo consequences in the context of cancer pathobiology.     

Opposite role of Ras in tumor necrosis factor-alpha-induced cell cycle regulation: competition for Raf kinase

We previously reported an epitope presenting vector, pCI, a derivative of a human invariant chain (Ii) expression vector, in which the class II associated invariant chain peptide (CLIP, Ii p89-101) could be substituted with antigenic peptides. In the current study, we used this vector to develop a new expression cloning system to identify CD4+ T cell epitopes. We inserted double-stranded oligo DNAs of randomized sequences into this vector and prepared an epitope-presenting library which loads randomized 13-mer peptides onto HLA class II molecules coexpressed in COS-7 cells. Utilizing this library, we isolated a cross-reactive epitope recognized by a glutamic acid decarboxylase (GAD) 65-autoreactive T cell clone established from a patient with insulin-dependent diabetes mellitus. Although the newly identified epitope (PVQLSNQWHVVGATF) was far different from the original epitope, GAD65 p116-128 (NILLQYVVKSFDR), it did have the capacity to stimulate the T cell clone comparable to that of the original GAD epitope. Our system may be applicable not only for identifying of cross-reactive epitopes for CD4+ T cells of known specificity, but also for detection of epitopes stimulatory for CD4+ T cells the epitopes of which are unknown.     

A consideration of the role of cell surface macromolecules in the process of viral transformation.

There is extensive physiological evidence implicating the cell surface as the key organelle which mediates the cell:cell interactions which underlie both normal and neoplastic growth. This information has now been supplemented with biochemical and biophysical data which indicates that surface macromolecules, in particular the heteroglycans of transformed cells, differ from those which lie at the periphery of normal cells. In the case of cells neoplastically transformed by most tumour viruses it is clear that the small virus genome (2-5 x 10(6) daltons) cannot carry the total genetic information to accomodate these various biochemical modifications, if indeed they are encoded in separate genes (1). To examine the part played in transformation by cellular genes coding for surface heteroglycan formation, we have turned to a study of SV-3T3 cells (ts H6-15) which are temperature-sensitive for expression of the transformed cell phenotype (2). The data show that cells grown under conditions permissive and non-permissive for such expression exhibit the same pattern of formation of glycolipids, and the majority of the polypeptides of the plasma membrane. There are, however, significant differences in the synthesis of some glycopeptides. A large molecular weight, trypsin-labile glycopeptide, present at the surface of untransformed fibroblasts but barely measurable in some of their virus-transformed derivatives (3), was detected, essentially at the same level, at the surface of ts H6-15 cells grown at the permissive and non-permissive temperatures. The signficance of these observations is discussed.     

Divide and conquer: development and cell cycle genes in plant transformation

Genetic transformation and regeneration of transgenic plants remains unfeasible for the majority of plant species. We propose that inducible expression and/or suppression of the genes that control the cell cycle and development, by altering chromatin structure and exerting epigenetic control of gene expression, might substantially improve competence for transformation and/or regeneration. Transformation efficiency was higher in cells with nuclei at the S and G2 phases, and manipulating the genes whose activation or silencing promote the G1-S transition has increased both transient and stable transformation. Controlling the cell cycle directly, using RBR and VIP1, or indirectly, through hormone regulation using IPT and ESR1, has improved rates of stable transformation. Other target genes that might promote incorporation of DNA and/or pluripotency of cells include HP1, CycD3 and CycD1. The availability of large EST databanks, complete plant-genome sequences and/or inducible gene expression systems create opportunities for testing homologous genes to increase competence of transformation and regeneration.     

Regulation of memory antibody levels: the role of persisting antigen versus plasma cell life span

Protective Ab levels can be maintained for years upon infection or vaccination. In this study, we studied the duration of Ab responses as a function of the life span of plasma cells and tested the role of persisting Ag in maintaining B cell memory. Our analysis of B cell responses induced in mice immunized with virus-like particles demonstrates the following: 1) Ab titers are long-lived, but decline continuously with a t(1/2) of approximately 80 days, which corresponds to the life span of plasma cells; 2) the germinal center (GC) reaction, which lasts for up to 100 days, is dependent on Ag associated with follicular dendritic cells; and 3) early GCs produce massive numbers of plasma and memory B cell precursors, whereas the late Ag-dependent GCs are dispensable for the maintenance of Ab levels and B cell memory.     

The role of lymphokine-activated cell-associated antigen. II. Distribution and correlation with cell cycle

It has previously been shown that killer-blocking monoclonal antibody (KBA MAb) recognizes lymphokine-activated cell-associated antigen (LAA) involved in broad-reactive killer. (BRK) cell-mediated cytotoxicity. We now report that LAA is expressed on all lymphoid cells, though the amount of LAA on unstimulated lymphocytes is low. In contrast, lymphocytes activated in vitro with either concanavalin A, alloantigens, lipopolysaccharide, or recombinant interleukin 2 express high levels of LAA. In addition, in vivo activated lymphocytes, such as OK-432-activated lymphocytes and tumor-infiltrating lymphocytes express higher levels of LAA than unstimulated lymphocytes. We also demonstrate that the expression of LAA is restricted in T-cell lymphomas and a M phi cell line, while myelomas, fibrosarcomas, and carcinomas do not express LAA. Cell cycle analysis using propidium iodide and KBA MAb showed that LAA expression was closely correlated with the transition of cells from G1a to G1b phase.     

Regulation of apoptosis and cell cycle progression by MCL1. Differential role of proliferating cell nuclear antigen

MCL1 (ML1 myeloid cell leukemia 1), a Bcl-2 (B- cell lymphoma-leukemia 2) homologue, is known to function as an anti-apoptotic protein. Here we show in vitro and in vivo that MCL1 interacts with the cell cycle regulator, proliferating cell nuclear antigen (PCNA). This finding prompted us to investigate whether MCL1, in addition to its anti-apoptotic function, has an effect on cell cycle progression. A bromodeoxyuridine uptake assay showed that the overexpression of MCL1 significantly inhibited the cell cycle progression through the S-phase. The S-phase of the cell cycle is also known to be regulated by PCNA. A mutant of MCL1 that lacks PCNA binding (MCL1(Delta)(4A)) could not inhibit cell cycle progression as effectively as wild type MCL1. In contrast, MCL1(Delta)(4A) retained its anti-apoptotic function in HeLa cells when challenged by Etoposide. In addition, the intracellular localization of MCL1(Delta)(4A) was identical to that of wild type MCL1. An in vitro pull-down assay suggested that MCL1 is the only Bcl-2 family protein to interact with PCNA. In fact, MCL1, not other Bcl-2 family proteins, contained the PCNA-binding motif described previously. Taken together, MCL1 is a regulator of both apoptosis and cell cycle progression, and the cell cycle regulatory function of MCL1 is mediated through its interaction with PCNA.     

Hepatitis C--new developments in the studies of the viral life cycle

Hepatitis C virus (HCV) is a causative agent of chronic liver disease leading to cirrhosis, liver failure and hepatocellular carcinoma. The prevalence of HCV is estimated as 3% of the world population and the virus is a major public health problem all over the world. For over 16 years, since HCV had been discovered, studies of the mechanisms of the viral life cycle and virus-host interactions have been hampered by the lack of a cell culture system allowing the virus to be grown in laboratory conditions. However, in recent years some new model systems to study HCV have been developed. The major breakthrough of the last two years was the cell culture system for maintaining the virus in an adapted hepatocyte-derived cell line. This review describes the techniques and applications of most of the in vitro systems and animal models currently used for working with hepatitis C virus.     

The role of serotonin in a bdelloid life cycle

Serotonin is a neurotransmitter present throughout animal kingdom that controls a number of crucial functions in different taxa. In this study we document the role of serotonin in a bdelloid rotifer, Macrotrachela quadricornifera. Following life table experimental protocol, we recorded age-specific fecundity and survival rates of cohorts that received WAY 100135 maleate, a selective antagonist of the serotonin receptor 1A. The antagonist was provided from the age of 4 days (age at first reproduction) until death to one cohort group, and during 15 days to another group. Both groups were cultivated regularly, and their life history traits were recorded. Treated rotifers continued to produce eggs, but most eggs were never oviposited and eventually ruptured the mother’s body wall. The retained eggs, being parthenogenetic began embryogenesis and some could hatch inside the mother’s body cavity (pseudocoel). When WAY was suspended, most treated rotifers oviposited all their eggs. About 75% of these eggs developed, but development was 1–2 days longer than in controls. Most newborns reached sexual maturity, although their maturity was delayed as well. Eggs produced after WAY suspension hatched as much as the controls (~100%). WAY possibly did not affect egg production, but prevented regular oviposition. Nevertheless, viability was reduced and development took longer during WAY treatment. Results suggest that in bdelloid rotifers serotonin is involved in the control of egg laying and development, among other features, as it does in most animals, from nematodes to chordates.