Translational control in virus-infected cells: models for cellular stress responses

Protein synthesis is regulated at the translational level by a variety of mechanisms in virus-infected cells. Viruses often induce the shut-off of host translation in order to favour the expression of their own genetic information, but cells possess a number of strategies for counteracting such effects of infection. Important regulatory mechanisms include the phosphorylation of the alpha subunit of polypeptide chain initiation factor eIF2, RNA degradation mediated by the 2'5'-oligoadenylate/RNase L system, control of availability of the cap-binding protein eIF4E by its interaction with the 4E-binding proteins and specific proteolytic cleavage of several key initiation factors. Most of these mechanisms are also utilised in uninfected cells in response to a variety of physiological stresses and during the early stages of apoptosis. Thus, mechanisms of translational control during virus infection can provide models for the cellular stress responses observed in a wide range of other circumstances.     

Methionine dependence of virus-infected cells

Mouse embryo cells nonproductively infected with human cytomegalovirus differed from noninfected cells by the impaired ability to grow in the medium containing homocysteine instead of methionine. Virus infection of mouse embryo cells grown in both kinds of media resulted in the increase of protein synthesis. In the infected cells grown on homocysteine this increase was followed by a quick decrease. The effects of homocysteine substitution could be abolished by the addition of low amounts of methionine (0.1 mM). Methionine uptake in the infected cells grown on homocysteine for 48 h was significantly higher than that in the noninfected cells.     

Viruses and interactomes in translation

A decade of high-throughput screenings for intraviral and virus-host protein-protein interactions led to the accumulation of data and to the development of theories on laws governing interactome organization for many viruses. We present here a computational analysis of intraviral protein networks (EBV, FLUAV, HCV, HSV-1, KSHV, SARS-CoV, VACV, and VZV) and virus-host protein networks (DENV, EBV, FLUAV, HCV, and VACV) from up-to-date interaction data, using various mathematical approaches. If intraviral networks seem to behave similarly, they are clearly different from the human interactome. Viral proteins target highly central human proteins, which are precisely the Achilles' heel of the human interactome. The intrinsic structural disorder is a distinctive feature of viral hubs in virus-host interactomes. Overlaps between virus-host data sets identify a core of human proteins involved in the cellular response to viral infection and in the viral capacity to hijack the cell machinery for viral replication. Host proteins that are strongly targeted by a virus seem to be particularly attractive for other viruses. Such protein-protein interaction networks and their analysis represent a powerful resource from a therapeutic perspective.     

Protein cleavage in virus-infected cells

A variety of proteins, including viral precursor polypeptides, were bound to a solid support and used in a sensitive assay for proteolytic enzymes in HeLa cells. A trypsin-like endoprotease, present on ribosomes of HeLa cells, loses activity after picornavirus infection. The decline follows synthesis and processing of a viral protein. Inhibition of cellfree activity of HeLa protease occurs when protein trypsin inhibitors or double-stranded RNA are added. After the mid-point of infection, protease activity with enhanced specificity for viral substrates is detected. The new protease has a pH optimum and heat stability different from endogenous host enzymes, and is synthesized following infection. A viral mutant was isolated which produces a temperature-sensitive protease. The results indicate that a poliovirus gene product participates enzymatically in the final cleavages of some polioviral proteins. A model for the regulation of poliovirus replication based on specific proteolysis is presented.     

Prediction and comparison of Salmonella-human and Salmonella-Arabidopsis interactomes

Salmonellosis caused by Salmonella bacteria is a food-borne disease and a worldwide health threat causing millions of infections and thousands of deaths every year. This pathogen infects an unusually broad range of host organisms including human and plants. A better understanding of the mechanisms of communication between Salmonella and its hosts requires identifying the interactions between Salmonella and host proteins. Protein-protein interactions (PPIs) are the fundamental building blocks of communication. Here, we utilize the prediction platform BIANA to obtain the putative Salmonella-human and Salmonella-Arabidopsis interactomes based on sequence and domain similarity to known PPIs. A gold standard list of Salmonella-host PPIs served to validate the quality of the human model. 24,726 and 10,926 PPIs comprising interactions between 38 and 33 Salmonella effectors and virulence factors with 9,740 human and 4,676 Arabidopsis proteins, respectively, were predicted. Putative hub proteins could be identified, and parallels between the two interactomes were discovered. This approach can provide insight into possible biological functions of so far uncharacterized proteins. The predicted interactions are available via a web interface which allows filtering of the database according to parameters provided by the user to narrow down the list of suspected interactions. The interactions are available via a web interface at http://sbi.imim.es/web/SHIPREC.php.     

Attachment characteristics of normal human cells and virus-infected cells on microcarriers

The attachment kinetics of normal and virus-infected LuMA cells were studied to improve the production of live attenuated varicella viruses in human embryonic lung (LuMA) cells. Normal LuMA cells and LuMA cells infected by varicella virus at various cytopathic effects (CPE) were grown on microcarriers. Ninety-three percent of suspended LuMA cells attached to the solid surface microcarriers within fifteen minutes and cell viability was greater than 95% when the cell suspension was stirred. Low serum levels did not affect the attachment rate of virus-infected cells in the microcarrier culture system. Kinetic studies showed that varicella infected cells had a lower attachment rate than normal LuMA cells. Virus inoculum (= infected cells) at low CPE showed a relatively better attachment rate on cell-laden microcarriers than virus inoculum at a higher CPE. Maximum titers were obtained at 2 days post-infection. Based on cell densities, the use of viral inoculum showing a 40% CPE led to an approximately 2- and 1.2-fold increase in the cell associated and in cell free viruses, respectively, than a virus inoculum with a CPE of 10%.However, the ratio of cell-free to cell-associated virus in a microcarrier culture was very low, approximately0.04–0.06. These studies demonstrate that the virus inoculum resulting in a high CPE yielded a high production of cell-associated and cell-free virus in microcarrier cultures because of the high cellular affinity of the varicella virus. This revised version was published online in August 2006 with corrections to the Cover Date.     

Nucleus-associated RNA in measles virus-infected cells

Measles virus RNA was found in the nuclear fraction of infected Vero cells. 24-hr labeling periods revealed heterogeneously sedimenting 15–50 S RNA associated with a membrane-containing particulate. Viral RNA isolated after shorter labeling periods was larger in size (30–50 S) and associated with both nucleoplasmic and particulate fractions.     

Translational control in influenza virus-infected cells

Influenza virus type A has been shown to establish a translational control system such that during infection there is a dramatic inhibition of host cell protein synthesis and viral mRNAs are selectively and efficiently translated. The following review summarizes the complex strategies employed by influenza to accomplish these goals. These include: (i) preventing newly made cellular mRNAs from entering the cytoplasm of infected cells; (ii) inhibiting the initiation and elongation steps of translation of preexisting cellular mRNAs; (iii) possessing RNAs with structural features which enhance translation; (iv) encoding mechanisms to downregulate the interferon induced protein kinase thus allowing overall protein synthesis levels to remain high.     

Charting plant interactomes: possibilities and challenges

Protein-protein interactions are essential for nearly all cellular processes. Therefore, an important goal of post-genomic research for defining gene function and understanding the function of macromolecular complexes involves creating 'interactome' maps from empirical or inferred datasets. Systematic efforts to conduct high-throughput surveys of protein-protein interactions in plants are needed to chart the complex and dynamic interaction networks that occur throughout plant development. However, no single approach can build a complete map of the interactome. Here, we review the utility and potential of various experimental approaches for creating large-scale protein-protein interaction maps in plants. Bioinformatics approaches for curating and assessing the confidence of these datasets through inter-species comparisons will be crucial in achieving a complete understanding of protein interaction networks in plants.     

Analysis of virus-infected cells by flow cytometry

Flow cytometry has been used to study virus-cell interactions for many years. This article critically reviews a number of reports on the use of flow cytometry for the detection of virus-infected cells directly in clinical samples and in virus-infected cultured cells. Examples are presented of the use of flow cytometry to screen antiviral drugs against human immunodeficiency virus (HIV), human cytomegalovirus, and herpes simplex viruses (HSV) and to perform drug susceptibility testing for these viruses. The use of reporter genes such as green fluorescent protein incorporated into HIV or HSV or into cells for the detection of the presence of virus, for drug susceptibility assay, and for viral pathogenesis is also covered. Finally, studies on the use of flow cytometry for studying the effect of virus infection on apoptosis and the cell cycle are summarized. It is hoped that this article will give the reader some understanding of the great potential of this technology for studying virus cell interactions.     

Polypeptide synthesis and phosphorylation in Epstein-Barr virus-infected cells

Epstein-Barr virus superinfection of the human lymphoblastoid cell line Raji, a Burkitt lymphoma-derived line that contains Epstein-Barr virus genomes in an episomal form, results in the sequential synthesis of 29 detectable proteins, which range in molecular weight from approximately 155,000 to 21,000, and in the shutoff of the bulk of host protein synthesis within 6 to 9 h after infection. There are three classes of virus-induced proteins; these are an early class, consisting of eight proteins synthesized by 6 h postinfection, an intermediate class, containing two proteins synthesized 9 h postinfection, and a late class, consisting of five proteins synthesized 12 h postinfection. In addition, there is a fourth class of polypeptides, called persistent, that are found both before and after superinfection. The rates of synthesis of the proteins fall into three patterns; these are pattern A, in which the rate of synthesis decreases, pattern B, in which the rate of synthesis remains steady, and pattern C, in which the rate of synthesis increases after the initial appearance of the polypeptide. Both 9-(2-hydroxy-ethoxymethyl)guanine (acyclovir) and phosphonoacetic acid inhibit the appearance of one intermediate protein and at least three late proteins. Seven polypeptides are phosphorylated at different times after infection.     

大规模蛋白质相互作用组实验技术及其应用

大规模蛋白质相互作用研究的主要实验技术包括酵母双杂交技术、串联亲和纯化技术和蛋白质芯片技术,随着这些技术的不断发展和完善,科学家们在模式生物、哺乳动物、病原微生物中展开了大规模的蛋白质相互作用组研究,并进行了药物研发方面的研究,绘制了多种生物的蛋白质相互作用连锁图,揭示了多种蛋白质的新功能,为全面研究蛋白质（群）的分子作用机制、药物研发和疾病的临床预防与治疗等提供了崭新的线索。