The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon

<p>Abstract</p> <p>Background</p> <p>Bovine viral diarrhea virus (BVDV) is the prototype representative of the pestivirus genus in the <it>Flaviviridae </it>family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA <abbrgrp><abbr bid="B1">1</abbr></abbrgrp>. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein <abbrgrp><abbr bid="B2">2</abbr></abbrgrp>. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV) and the more distantly related Hepatitis C virus (HCV) pseudoknot function in translation has been demonstrated.</p> <p>Results</p> <p>To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical swine fever virus, a pestivirus, and the hepatitis C viruses, another genus of the <it>Flaviviridae</it>.</p> <p>Conclusion</p> <p>The IRES of the non-cytopathic BVDV SD-1 strain displays features known from other pestivirus IRESes. The predicted pseudoknot in the 5'UTR of BVDV SD-1 virus represents an important structural element in BVDV translation.</p

Physical and mathematical justification of the numerical Brillouin zone integration of the Boltzmann rate equation by Gaussian smearing

Scatterings of electrons at quasiparticles or photons are very important for many topics in solid state physics, e.g., spintronics, magnonics or photonics, and therefore a correct numerical treatment of these scatterings is very important. For a quantum-mechanical description of these scatterings Fermi's golden rule is used in order to calculate the transition rate from an initial state to a final state in a first-order time-dependent perturbation theory. One can calculate the total transition rate from all initial states to all final states with Boltzmann rate equations involving Brillouin zone integrations. The numerical treatment of these integrations on a finite grid is often done via a replacement of the Dirac delta distribution by a Gaussian. The Dirac delta distribution appears in Fermi's golden rule where it describes the energy conservation among the interacting particles. Since the Dirac delta distribution is a not a function it is not clear from a mathematical point of view that this procedure is justified. We show with physical and mathematical arguments that this numerical procedure is in general correct, and we comment on critical points

Amphotropic murine leukaemia virus envelope protein is associated with cholesterol-rich microdomains

BACKGROUND: Cholesterol-rich microdomains like lipid rafts were recently identified as regions within the plasma membrane, which play an important role in the assembly and budding of different viruses, e.g., measles virus and human immunodeficiency virus. For these viruses association of newly synthesized viral proteins with lipid rafts has been shown. RESULTS: Here we provide evidence for the association of the envelope protein (Env) of the 4070A isolate of amphotropic murine leukaemia virus (A-MLV) with lipid rafts. Using density gradient centrifugation and immunocytochemical analyses, we show that Env co-localizes with cholesterol, ganglioside GM1 and caveolin-1 in these specific regions of the plasma membrane. CONCLUSIONS: These results show that a large amount of A-MLV Env is associated with lipid rafts and suggest that cholesterol-rich microdomains are used as portals for the exit of A-MLV

Caveolin-1 influences human influenza A virus (H1N1) multiplication in cell culture

<p>Abstract</p> <p>Background</p> <p>The threat of recurring influenza pandemics caused by new viral strains and the occurrence of escape mutants necessitate the search for potent therapeutic targets. The dependence of viruses on cellular factors provides a weak-spot in the viral multiplication strategy and a means to interfere with viral multiplication.</p> <p>Results</p> <p>Using a motif-based search strategy for antiviral targets we identified caveolin-1 (Cav-1) as a putative cellular interaction partner of human influenza A viruses, including the pandemic influenza A virus (H1N1) strains of swine origin circulating from spring 2009 on. The influence of Cav-1 on human influenza A/PR/8/34 (H1N1) virus replication was determined in inhibition and competition experiments. RNAi-mediated Cav-1 knock-down as well as transfection of a dominant-negative Cav-1 mutant results in a decrease in virus titre in infected Madin-Darby canine kidney cells (MDCK), a cell line commonly used in basic influenza research as well as in virus vaccine production. To understand the molecular basis of the phenomenon we focussed on the putative caveolin-1 binding domain (CBD) located in the lumenal, juxtamembranal portion of the M2 matrix protein which has been identified in the motif-based search. Pull-down assays and co-immunoprecipitation experiments showed that caveolin-1 binds to M2. The data suggest, that Cav-1 modulates influenza virus A replication presumably based on M2/Cav-1 interaction.</p> <p>Conclusion</p> <p>As Cav-1 is involved in the human influenza A virus life cycle, the multifunctional protein and its interaction with M2 protein of human influenza A viruses represent a promising starting point for the search for antiviral agents.</p

Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production

BACKGROUND: Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. RESULTS: The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV) at the plasma membrane (PM) and the formation of virus like particles in multivesicular bodies (MVBs). In our study we show that caveolin-1 (Cav-1), a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA) of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD) within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other γ-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. CONCLUSION: This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly

Nutrient Composition of some Tropical Legumes Capable of Substituting Fish Meal in Fish Diets

Sword beans (Canavalia gladiata); Jackbean (Canavalia enciformis (L)); Mucuna bean (Mucuna pruriens); Mucuna cochiunensis; Bambara (Voandzeia subterranea) and Limabean (Phaseolus lunatus) are the tropical legumes considered in this paper. They have been used in the feed of ruminants but very scarcely considered in fish feed. Information about their nutrient composition are also scarce. Results from this study show that the protein contents of the test seeds ranged from 19.94% dry matter (DM), (Bambara) to 36.95% DM (Mucuna cochiunensis). Considering the high protein level required by fish for maximum growth and the presence of some ANFs, the seeds may not be able to be used in isolation without supplementing them with other food stuffs having higher protein value. The relatively high content of Nitrogen Free Extract (+ fibre) seem to suggest that the test seeds can be used in a semi-intensive setting to supply carbohydrate in fish diets. The seeds contain considerable amount of linoleic acid (18:2 n-6). The highest occurring in Lima beans. Sword beans and Jack beans are rich in oleic acid (18:1n-9). Palmatic acid (16:0) is high, while stearic acid (18:0) and myristic acid (14:0) are low. The amino acid compositions of the test seeds are not very adequate. Sword beans had a better amino acid profile though it seems deficient in some of the amino acids. The amino acid contents of Jack bean, Mucuna bean, Bambara and Lima bean look inadequate to provide a possible alternative to fish meal on individual basis. If to be used in fish feed formulation, combinating them with other protein sources, possessing higher contents of the limiting amino acids is strongly suggested. The potentials of these seeds in fish feed formulation seem high

Pharmacological Modulation of Mucosa-Related Impairment of β-Adrenoceptor-Mediated Relaxation in Human Detrusor

Objectives: The mucosa of human detrusor strips impairs catecholamine-induced relaxation. In order to elucidate which signal transduction pathways are involved in this cross talk between the mucosa and detrusor, we have studied the effects of several pharmacological agonists and antagonists on noradrenaline-mediated relaxation in intact and mucosa-denuded detrusor strips. Patients and Methods: Strips of detrusor tissue were obtained from patients who had undergone cystectomy for bladder cancer and were set up for force measurement. KCl- or carbachol-precontracted strips were relaxed with increasing concentrations of noradrenaline in the absence and in the presence of nitric oxide synthase inhibitor, L-NAME; P2X-receptor antagonist, PPADS; ET A -receptor antagonist, BQ-123; ET B -receptor antagonist, BQ-788; cyclooxygenase inhibitor, diclofenac; AT 1 -receptor antagonist, candesartan; and NK 1 -receptor antagonist, L-703,606. Results: In intact strips, KCl-stimulated force was enhanced by all blockers; carbachol-stimulated force increased with L-703,606. In denuded strips, only L-NAME augmented the KCl-stimulated contraction. Noradrenaline relaxed the precontracted detrusor strips to a significantly larger extent and at lower concentrations in denuded than in intact strips. L-NAME, PPADS and BQ-123/BQ-788 had little effect on noradrenaline-induced relaxation, whereas diclofenac, candesartan and L-703,606 sensitized intact carbachol-stimulated detrusor strips to noradrenaline-induced relaxation. Conclusion: Inhibition of the noradrenaline-induced relaxation of precontracted human detrusor strips by the mucosa is attenuated by diclofenac, candesartan and L-703,606 suggesting the involvement of prostanoids, angiotensin and neurokinin pathways. Further experiments are required to unravel the exact mechanisms

Influence of dietary protein deficiency on amino acid and fatty acid composition in tilapia, Oreochromis niloticus, fingerlings.

Abstract The influence of dietary protein deficiency on the amino acid and fatty acid compositions of tilapia (Oreochromis niloticus) fingerlings was studied. Two experimental diets (0.81% and 33.32% protein, dry matter) were prepared. The protein content of fish fed diet 1 (0.81% protein) decreased from 57.14% to 49.18% in eight weeks. Fish fed diet 2 (33.32% protein) had higher protein and amino acid contents. The lipid content of fish fed diet 1 was higher than that of fish fed diet 2, suggesting that carbohydrates transformed into lipids. The levels of fatty acids 16:0 and 18:2 n-6 in fish fed diet 1 remained nearly unchanged and did not reflect the diet, demonstrating that fatty acids in diet 1 may not have been incorporated into the triglycerides of the tissues. Possible impairment of lipid secretion from the liver, caused by depletion of protein in the blood lipoprotein, may have affected the transport of lipids to the muscles. A dietary protein deficiency results not only in a deficiency of essential amino acids in the body but also affects transport and storage of lipids within the fish