The molecular chaperone heat shock protein-90 positively regulates rotavirus infection

AbstractRotaviruses are the major cause of severe dehydrating gastroenteritis in children worldwide. In this study, we report a positive role of cellular chaperone Hsp90 during rotavirus infection. A highly specific Hsp90 inhibitor, 17-allylamono-demethoxygeldanamycin (17-AAG) was used to delineate the functional role of Hsp90. In MA104 cells treated with 17-AAG after viral adsorption, replication of simian (SA11) or human (KU) strains was attenuated as assessed by quantitating both plaque forming units and expression of viral genes. Phosphorylation of Akt and NFκB observed 2–4 hpi with SA11, was strongly inhibited in the presence of 17-AAG. Direct Hsp90–Akt interaction in virus infected cells was also reduced in the presence of 17-AAG. Anti-rotaviral effects of 17-AAG were due to inhibition of activation of Akt that was confirmed since, PI3K/Akt inhibitors attenuated rotavirus growth significantly. Thus, Hsp90 regulates rotavirus by modulating cellular signaling proteins. The results highlight the importance of cellular proteins during rotavirus infection and the possibility of targeting cellular chaperones for developing new anti-rotaviral strategies

RNA-controlled nucleocytoplasmic shuttling of mRNA decay factors regulates mRNA synthesis and a novel mRNA decay pathway

mRNA level is controlled by factors that mediate both mRNA synthesis and decay, including the 5' to 3' exonuclease Xrn1. Here we show that nucleocytoplasmic shuttling of several yeast mRNA decay factors plays a key role in determining both mRNA synthesis and decay. Shuttling is regulated by RNAcontrolled binding of the karyopherin Kap120 to two nuclear localization sequences (NLSs) in Xrn1, location of one ofwhich is conserved fromyeast to human. The decaying RNA binds and masks NLS1, establishing a link between mRNA decay and Xrn1 shuttling. Preventing Xrn1 import, either by deleting KAP120 or mutating the two Xrn1 NLSs, compromises transcription and, unexpectedly, also cytoplasmic decay, uncovering a cytoplasmic decay pathway that initiates in the nucleus.MostmRNAs are degraded by both pathways - the ratio between them represents a full spectrum. Importantly, Xrn1 shuttling is required for proper responses to environmental changes, e.g., fluctuating temperatures, involving proper changes in mRNA abundance and in cell proliferation rate

<em>In Silico</em> Study of Rotavirus VP7 Surface Accessible Conserved Regions for Antiviral Drug/Vaccine Design

<div><h3>Background</h3><p>Rotaviral diarrhoea kills about half a million children annually in developing countries and accounts for one third of diarrhea related hospitalizations. Drugs and vaccines against the rotavirus are handicapped, as in all viral diseases, by the rapid mutational changes that take place in the DNA and protein sequences rendering most of these ineffective. As of now only two vaccines are licensed and approved by the WHO (World Health Organization), but display reduced efficiencies in the underdeveloped countries where the disease is more prevalent. We approached this issue by trying to identify regions of surface exposed conserved segments on the surface glycoproteins of the virion, which may then be targeted by specific peptide vaccines. We had developed a bioinformatics protocol for these kinds of problems with reference to the influenza neuraminidase protein, which we have refined and expanded to analyze the rotavirus issue.</p> <h3>Results</h3><p>Our analysis of 433 VP7 (Viral Protein 7 from rotavirus) surface protein sequences across 17 subtypes encompassing mammalian hosts using a 20D Graphical Representation and Numerical Characterization method, identified four possible highly conserved peptide segments. Solvent accessibility prediction servers were used to identify that these are predominantly surface situated. These regions analyzed through selected epitope prediction servers for their epitopic properties towards possible T-cell and B-cell activation showed good results as epitopic candidates (only dry lab confirmation).</p> <h3>Conclusions</h3><p>The main reasons for the development of alternative vaccine strategies for the rotavirus are the failure of current vaccines and high production costs that inhibit their application in developing countries. We expect that it would be possible to use the protein surface exposed regions identified in our study as targets for peptide vaccines and drug designs for stable immunity against divergent strains of the rotavirus. Though this study is fully dependent on computational prediction algorithms, it provides a platform for wet lab experiments.</p> </div

MHC-II binding results for the identified four peptide segments.

<p>Results of the peptide-a, b, c and d submitted to the IEDB T-cell MHC-II binding prediction server are tabulated here. For each peptide the server generates two overlapping peptides; the peptides for the four groups are shown in different colors.</p

Position of Peptide-b is shown from outer surface of the virion in 3D-space filling model of rotaviral trimeric VP7.

<p>Peptide-b (aa 242 to aa 255) is shown in blue color and clearly highlights its discontinuous characteristic.</p

Comparison of solvent accessibility and stretch variability in rotaviral VP7 sequence database.

<p>Comparative diagram between solvent accessibility and stretch variability shows the regions of lowest variability and greatest environmentally accessed. All nine variable regions documented from previous research are found clearly distinguishable through our graph. Those regions are marked in green line. Peptide regions (peptide-a, b, c & d) indentified by our method are marked by blue lines.</p

Assignment of axis to individual amino acids.

<p>Assignment of axis to individual amino acids.</p

Position of Peptide-b is shown from inner surface of the virion in 3D-space filling model of rotaviral trimeric VP7.

<p>Lower part of Peptide-b (aa 242 to aa 255) is shown in blue color. It’s the same blue peptide as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040749#pone-0040749-g005" target="_blank">Figure 5</a>.</p