Human leukocyte interferon subtypes have different antiproliferative and antiviral activities on human cells

The antigrowth effects of 5 different cloned human leukocyte IFN subtypes (IFN-alpha A, B, C, D, F) and 2 molecular hybrids between them (IFN-alpha AD(Bg1II) and IFN-alpha DA(Bg1II)) were examined on 6 different human cell lines. The results indicate that the interferons sort into two distinct groups: IFN-alpha B, C and F showed comparable antiproliferative activity which was greater than that of IFN-alpha A, D, AD(Bg1II) and DA(Bg1II). The interferons could also be assigned to one of two groups on the basis of their antiviral activity. IFN-alpha A, D and AD(Bg1II) were observed to be more protective than IFN-alpha B, C and F against HSV-2 and EMCV infections, i.e. the relative antiviral efficacies of the cloned IFN subtypes were the reverse of their antiproliferative activities.     

Comparison of impact of EMCV replication on the nuclear apparatus of NIH 3T3 and HEp-2 cells

We have investigated differences between the actions of encephalomyocarditis virus (EMCV) on cytometric indices in cultured NIH 3T3 and HEp-2 cells, which are characterized by different levels of transformation. HEp-2 cells surviving 48 h after EMCV infection showed lower nuclear ploidy, reduced nuclear area, fewer nucleoli and a higher percentage of euploid cells. There was a significant increase of nucleolar/nuclear DNA 6-24 h after EMCV infection. However, EMCV had markedly different effects on NIH 3T3 cells: there was a consistent increase in population ploidy, but the average number of nucleoli and the number of euploid cells in the population remained constant. The nucleolar/nuclear DNA ratio was almost unchanged. These different viral effects might be explained by the contrasting levels of differentiation of the cultured cell lines. The number of nucleoli does not depend on the amount of nuclear DNA in either viral-infected or intact cells but on the euploidy-to-aneuploidy ratio. The ratio of the sums of the nucleolar perimeters to the nuclear perimeter increases linearly with the number of nucleoli per nucleus in both intact and virus-infected cells. In both cell lines, the amount of DNA per nucleolus decreases as the number of nucleoli increases.     

Effects of magnesium ions on two EMCV IRES key activity fragments

Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino protons, changes have been monitored in base-pair kinetics induced by Mg^2 + in two key activity fragments r(CACCUGGCGACAGGUG) and r(GGCCAAAAGCC) of the encephalomyocarditis virus internal ribosome entry site. For r(CACCUGGCGACAGGUG), the addition of Mg^2 + reveals two types of base-pairs: r(U⁵⁴⁵·A) and r(G⁵⁴⁶·C), in the first category, have lifetimes only slightly higher in the presence of Mg^2 +, whereas their dissociation constants are substantially reduced. This behavior has been termed proximal. The base-pairs r(G⁵⁵³·C) and r(G⁵⁵⁴·C), in the second category, have lifetimes substantially higher in the presence of Mg^2 +, whereas their dissociation constants remain almost constant. This behavior has been termed distal. Mg^2 + has a specific effect on r(CACCUGGCGACAGGUG), the magnitude of which is progressively modulated from the proximal region of the 16-mer towards its distal region. For r(GGCCAAAAGCC), an intermediate behavior is found for base-pairs r(G⁵⁶⁵·C) and r(G⁵⁷²·C). Their lifetimes are slightly higher in the presence of Mg^2 + and their dissociation constants are significantly lower, a behavior resembling that of the 16-mer proximal region. These results indicate that Mg^2 + diffusively moves around r(GGCCAAAAGCC).     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.     

Oncostatin M synergistically inhibits HCV RNA replication in combination with interferon-α

Oncostatin M (OSM), a member of the interleukin-6 family, possesses various functions, including hepatocyte differentiation and suppression of melanoma cell growth. Here, we report anti-hepatitis C virus (HCV) activity of OSM as a new function of this cytokine. OSM possessed marked anti-HCV activity (50% effective concentration: 0.71 ng/ml) in an HCV RNA replication cell culture system. The most striking finding is that OSM exhibited synergistic inhibitory activity on interferon (IFN)-α even at a low concentration with weak anti-HCV activity, such as 25 pg/ml. OSM is a candidate anti-HCV reagent and may improve the current IFN therapy for patients with chronic hepatitis C.     

Faulty old ideas about translational regulation paved the way for current confusion about how microRNAs function

Despite a recent surge of reports about how microRNAs might regulate translation, the question has not been answered. The proposed mechanisms contradict one another, and none is supported by strong evidence. This review explains some deficiencies in the experiments with microRNAs. Some of the problems are traceable to bad habits carried over from older studies of translational regulation, here illustrated by discussing two models involving mRNA binding proteins. One widely-accepted model, called into doubt by recent findings, is the maskin hypothesis for translational repression of cyclin B1 in Xenopus oocytes. The second dubious model postulates repression of translation of ceruloplasmin by mRNA binding proteins. A big fault in the latter case is reconstructing the imagined mechanism before looking carefully at the real thing--a criticism that applies also to studies with microRNAs. Experiments with microRNAs often employ internal ribosome entry sequences (IRESs) as tools, necessitating brief discussion of that topic. A sensitive new assay reveals that many putative IRESs promote expression of downstream cistrons via splicing rather than internal initiation of translation. Recent claims about the biological importance of IRES-binding proteins--including suggestions that these proteins might serve as targets for cancer therapy--are not supported by any meaningful evidence. The bottom line is that older studies of mRNA binding proteins and putative IRESs have created a confusing picture of translational regulation which is not helpful when trying to understand how microRNAs might work. The obvious biological importance of microRNAs makes it essential to understand how they do what they do. Fresh ways of thinking and looking are needed.     

NMD resulting from encephalomyocarditis virus IRES-directed translation initiation seems to be restricted to CBP80/20-bound mRNA

Nonsense-mediated messenger RNA decay (NMD) generally degrades mRNAs that prematurely terminate translation as a means of quality control. NMD in mammalian cells targets newly spliced mRNA that is bound by the cap-binding protein heterodimer CBP80/20 and one or more post-splicing exon junction complexes during a pioneer round of translation. NMD targets mRNA that initiates translation using the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), therefore NMD might target not only CBP80/20-bound mRNA but also its remodelled product, eIF4E-bound mRNA. Here, we provide evidence that NMD triggered by translation initiation at the EMCV IRES, similar to NMD triggered by translation initiation at an mRNA cap, targets CBP80/20-bound mRNA but does not detectably target eIF4E-bound mRNA. We show that EMCV IRES-initiated translation undergoes a CBP80/20-associated pioneer round of translation that results in CBP80/20-dependent and Upf factor-dependent NMD when translation terminates prematurely.     

关于脑心肌炎病毒2A蛋白的研究进展

脑心肌炎病毒(Encephalomyocarditis virus,EMCV)是一种无囊膜的单股RNA病毒,属于小RNA病毒科,能够引起多种哺乳动物乃至人的感染。其非结构蛋白2A是重要的毒力因子,能够通过阻断翻译起始复合物的形成、结合翻译起始复合物因子及核糖体40s小亚基等方式竞争性地抑制宿主细胞蛋白的合成,还可通过抑制宿主细胞凋亡促进病毒扩散,并通过激活NF-κB引起宿主发生强烈的炎症反应[1]。此外,根据EMCV 2A蛋白的生物学特性,近年来,细胞生物学、病毒学领域均将其作为真核细胞与病毒互作的生物学工具展开了深入研究。