Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-beta and TNF-alpha production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E) from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP). In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-alpha as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-alpha and IFN-beta as well as the dsRNA analog, poly (I:C). Both IFN-beta and TNF-alpha further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.

HCV-like IRESs sequester eIF3: advantage virus

In a recent Nature paper, Hashem et al. attempted to probe deeper into the elusive role of eIF3 in translation initiation of viruses with hepatitis C virus-like internal ribosome entry sites (IRESs), but instead uncovered a surprising role of these IRESs in displacing eIF3 from the 40S subunit, favoring viral translation.

The N-terminal region containing the zinc finger domain of tobacco streak virus coat protein is essential for the formation of virus-like particles

Tobacco streak virus (TSV), a member of the genus Ilarvirus (family Bromoviridae), has a tripartite genome and forms quasi-isometric virions. All three viral capsids, encapsidating RNA 1, RNA 2 or RNA 3 and subgenomic RNA 4, are constituted of a single species of coat protein (CP). Formation of virus-like particles (VLPs) could be observed when the TSV CP gene was cloned and the recombinant CP (rCP) was expressed in E. coli. TSV VLPs were found to be stabilized by Zn2+ ions and could be disassembled in the presence of 500 mM CaCl2. Mutational analysis corroborated previous studies that showed that an N-terminal arginine-rich motif was crucial for RNA binding; however, the results presented here demonstrate that the presence of RNA is not a prerequisite for assembly of TSV VLPs. Instead, the N-terminal region containing the zinc finger domain preceding the arginine-rich motif is essential for assembly of these VLPs.

Emergent properties of the interferon-signalling network may underlie the success of hepatitis C treatment

Current interferon alpha-based treatment of hepatitis C virus (HCV) infection fails to cure a sizeable fraction of patients treated. The cause of this treatment failure remains unknown. Here using mathematical modelling, we predict treatment failure to be a consequence of the emergent properties of the interferon-signalling network. HCV induces bistability in the network, creating a new steady state where it can persist. Cells that admit the new steady state alone are refractory to interferon. Using a model of viral kinetics, we show that when the fraction of cells refractory to interferon in a patient exceeds a critical value, treatment fails. Direct-acting antivirals that suppress HCV replication can eliminate the new steady state, restoring interferon sensitivity and improving treatment response. Our study thus presents a new conceptual basis of HCV persistence and treatment response, elucidates the origin of the synergy between interferon and direct-acting antivirals, and facilitates rational treatment optimization.

Groundnut Bud Necrosis Virus Encoded NSm Associates with Membranes via Its C-Terminal Domain

Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200-250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell.

Virus-sized colloid transport in a single pore: Model development and sensitivity analysis

A mathematical model is developed to simulate the transport and deposition of virus-sized colloids in a cylindrical pore throat considering various processes such as advection, diffusion, colloid-collector surface interactions and hydrodynamic wall effects. The pore space is divided into three different regions, namely, bulk, diffusion and potential regions, based on the dominant processes acting in each of these regions. In the bulk region, colloid transport is governed by advection and diffusion whereas in the diffusion region, colloid mobility due to diffusion is retarded by hydrodynamic wall effects. Colloid-collector interaction forces dominate the transport in the potential region where colloid deposition occurs. The governing equations are non-dimensionalized and solved numerically. A sensitivity analysis indicates that the virus-sized colloid transport and deposition is significantly affected by various pore-scale parameters such as the surface potentials on colloid and collector, ionic strength of the solution, flow velocity, pore size and colloid size. The adsorbed concentration and hence, the favorability of the surface for adsorption increases with: (i) decreasing magnitude and ratio of surface potentials on colloid and collector, (ii) increasing ionic strength and (iii) increasing pore radius. The adsorbed concentration increases with increasing Pe, reaching a maximum value at Pe = 0.1 and then decreases thereafter. Also, the colloid size significantly affects particle deposition with the adsorbed concentration increasing with increasing particle radius, reaching a maximum value at a particle radius of 100 nm and then decreasing with increasing radius. System hydrodynamics is found to have a greater effect on larger particles than on smaller ones. The secondary minimum contribution to particle deposition has been found to increase as the favorability of the surface for adsorption decreases. The sensitivity of the model to a given parameter will be high if the conditions are favorable for adsorption. The results agree qualitatively with the column-scale experimental observations available in the literature. The current model forms the building block in upscaling colloid transport from pore scale to Darcy scale using Pore-Network Modeling. (C) 2014 Elsevier By. All rights reserved.

Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NF kappa B and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NF kappa B dependent since it was blocked by inhibitors of NF kappa B activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NF kappa B inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-alpha was also inhibited by ShRNA mediated knockdown of NF kappa B. TNF-alpha treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NF kappa B-mediated induction of HLA-F following JEV infection and TNF-alpha exposure is being suggested for the first time. (C) 2014 Elsevier Inc. All rights reserved.

Humanized antibody neutralizing 2009 pandemic H1N1 virus

The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of influenza virus neutralizing monoclonal antibodies (MAbs) for treatment purposes is a viable alternative. We previously reported the isolation of a high affinity, potently neutralizing murine MAb MA2077 against 2009 pandemic H1N1 virus. We describe here the humanization of MA2077 and its expression in a mammalian cell line. Six complementarity-determining regions (CDRs) of MA2077 were grafted onto the human germline variable regions; along with six and eight back mutations in the framework of heavy and light chains, respectively, pertaining to the vernier zone and interchain packing residues to promote favorable CDR conformation and facilitate antigen binding. The full length humanized antibody, 2077Hu2, expressed in CHO-K1 cells, showed high affinity to hemagglutinin protein (K-D = 0.75 +/- 0.32 nM) and potent neutralization of pandemic H1N1 virus (IC50 = 0.17 mu g/mL), with marginally higher IC50 as compared to MA2077 (0.08 mu g/mL). In addition, 2077Hu2 also retained the epitope specificity for the ``Sa'' antigenic site on pandemic HA. To the best of our knowledge, this is the first report of a humanized neutralizing antibody against pandemic H1N1 virus.

Adaptive evolution of a novel avian-origin influenza A/H7N9 virus

In China, the recent outbreak of novel influenza A/H7N9 virus has been assumed to be severe, and it may possibly turn brutal in the near future. In order to develop highly protective vaccines and drugs for the A/H7N9 virus, it is critical to find out the selection pressure of each amino acid site. In the present study, six different statistical methods consisting of four independent codon-based maximum likelihood (CML) methods, one hierarchical Bayesian (HB) method and one branch-site (BS) method, were employed to determine if each amino acid site of A/H7N9 virus is under natural selection pressure. Functions for both positively and negatively selected sites were inferred by annotating these sites with experimentally verified amino acid sites. Comprehensively, the single amino acid site 627 of PB2 protein was inferred as positively selected and it function was identified as a T-cell epitope (TCE). Among the 26 negatively selected amino acid sites of PB2, PB1, PA, HA, NP, NA, M1 and NS2 proteins, only 16 amino acid sites were identified to be involved in TCEs. In addition, 7 amino acid sites including, 608 and 609 of PA, 480 of NP, and 24, 25, 109 and 205 of M1, were identified to be involved in both B-cell epitopes (BCEs) and TCEs. Conversely, the function of positions 62 of PA, and, 43 and 113 of HA was unknown. In conclusion, the seven amino acid sites engaged in both BCEs and TCEs were identified as highly suitable targets, as these sites will be predicted to play a principal role in inducing strong humoral and cellular immune responses against A/H7N9 virus. (C) 2014 Elsevier Inc. All rights reserved.

Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells

Productive infection of human endothelial cells with Japanese encephalitis virus (JEV), a single stranded RNA virus induces shedding of sHLA-E. We show here that sHLA-E that is released upon infection with this flavivirus can inhibit IL-2 and PMA mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. Virus infected or IFN-gamma treated cell culture supernatants containing sHLA-E were found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. It was also found that sHLA-E could inhibit IL-2 induced H-3]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit NK cell responses. Hence JEV-induced shedding of sHLA-E needs further investigation to better understand immune responses in JEV infections since it may have a role in viral evasion of NK cell responses. (C) 2014 Elsevier B.V. All rights reserved.

Measuring Glutathione Redox Potential of HIV-1-infected Macrophages

Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E-GSH; Grx1-roGFP2) and measured subcellular changes in E-GSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E-GSH (approximately -310 mV), active viral replication induces substantial oxidative stress (E-GSH more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E-GSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about similar to 25 mV in E-GSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E-GSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV.

Demonstration of helicase activity in the nonstructural protein, NSs, of the negative-sense RNA virus, Groundnut bud necrosis virus

The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 50 a phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.

Viruses and viral proteins

For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes.

Fault Tolerance on Large Scale Systems using Adaptive Process Replication

Exascale systems of the future are predicted to have mean time between failures (MTBF) of less than one hour. At such low MTBFs, employing periodic checkpointing alone will result in low efficiency because of the high number of application failures resulting in large amount of lost work due to rollbacks. In such scenarios, it is highly necessary to have proactive fault tolerance mechanisms that can help avoid significant number of failures. In this work, we have developed a mechanism for proactive fault tolerance using partial replication of a set of application processes. Our fault tolerance framework adaptively changes the set of replicated processes periodically based on failure predictions to avoid failures. We have developed an MPI prototype implementation, PAREP-MPI that allows changing the replica set. We have shown that our strategy involving adaptive process replication significantly outperforms existing mechanisms providing up to 20 percent improvement in application efficiency even for exascale systems.

Targeted Modifications in Adeno-Associated Virus Serotype 8 Capsid Improves Its Hepatic Gene Transfer Efficiency In Vivo

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T -> Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S -> A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (similar to 9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h. FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h. FIX: Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.