ABSTRACT Human parainfluenza virus type 3 (HPIV3) is a negative-sense single-stranded RNA virus belonging to the Paramyxoviridae family. HPIV3 is a lung-tropic virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. The activation of the inflammasome by pathogens results in the production of proinflammatory cytokines such as interleukin-1β (IL-1β) during infection. Thus, the inflammasome-mediated proinflammatory response plays a critical role in regulating the immune response and virus clearance. The inflammasome is a multimeric protein complex triggering caspase-1 activation. Activated caspase-1 cleaves pro-IL-1β into its mature (and active) secretory form. Our study revealed inflammasome activation in macrophages following HPIV3 infection. Specifically, the activation of the NLRP3/ASC inflammasome resulted in the production of mature IL-1β from HPIV3-infected cells. Furthermore, Toll-like receptor 2 (TLR2) activation (first signal) and potassium efflux (second signal) constituted two cellular events mediating inflammasome activation following HPIV3 infection. During our studies, we surprisingly identified the HPIV3 C protein as an antagonist of inflammasome activation. The HPIV3 C protein is an accessory protein encoded by the open reading frame of the viral phosphoprotein (P) gene. The HPIV3 C protein interacted with the NLRP3 protein and blocked inflammasome activation by promoting the proteasomal degradation of the NLRP3 protein. Thus, our studies report NLRP3/ASC inflammasome activation by HPIV3 via TLR2 signaling and potassium efflux. Furthermore, we have identified HPIV3 C as a viral component involved in antagonizing inflammasome activation. IMPORTANCE Human parainfluenza virus type 3 (HPIV3) is a paramyxovirus that causes respiratory tract diseases during infancy and childhood. Currently, there is no effective vaccine or antiviral therapy for HPIV3. Therefore, in order to develop anti-HPIV3 agents (therapeutics and vaccines), it is important to study the HPIV3-host interaction during the immune response. Inflammasomes play an important role in the immune response. Inflammasome activation by HPIV3 has not been previously reported. Our studies demonstrated inflammasome activation by HPIV3 in macrophages. Specifically, HPIV3 activated the NLRP3/ASC inflammasome by TLR2 activation and potassium efflux. C proteins of paramyxoviruses are accessory proteins encoded by the viral phosphoprotein gene. The role of the C protein in inflammasome regulation was unknown. Surprisingly, our studies revealed that the HPIV3 C protein antagonizes inflammasome activation. In addition, we highlighted for the first time a mechanism utilized by paramyxovirus accessory proteins to block inflammasome activation. The HPIV3 C protein interacted with the NLRP3 protein to trigger the proteasomal degradation of the NLRP3 protein.

Event-by-event fluctuations of the multiplicities of inclusive charged particles and photons at forward rapidity in Au + Au collisions at root s(NN) = 200 GeV have been studied. The dominant contribution to such fluctuations is expected to come from correlated production of charged and neutral pions. We search for evidence of dynamical fluctuations of different physical origins. Observables constructed out of moments of multiplicities are used as measures of fluctuations. Mixed events and model calculations are used as base lines. Results are compared to the dynamical net-charge fluctuations measured in the same acceptance. A nonzero statistically significant signal of dynamical fluctuations is observed in excess to the model prediction when charged particles and photons are measured in the same acceptance. We find that, unlike dynamical net-charge fluctuation, charge-neutral fluctuation is not dominated by correlation owing to particle decay. Results are compared to the expectations based on the generic production mechanism of pions owing to isospin symmetry, for which no significant (<1%) deviation is observed.

We report on measurements of dielectron (e(+) e(-)) production in Au + Au collisions at a center-of-mass energy of 200 GeV per nucleon-nucleon pair using the STAR detector at BNL Relativistic Heavy Ion Collider. Systematic measurements of the dielectron yield as a function of transverse momentum (p(T)) and collision centrality show an enhancement compared to a cocktail simulation of hadronic sources in the low invariant-mass region (M-ee < 1 GeV / c(2)). This enhancement cannot be reproduced by the rho-meson vacuum spectral function. In minimum-bias collisions, in the invariant-mass range of 0.30-0.76 GeV / c(2), integrated over the full pT acceptance, the enhancement factor is 1.76 +/- 0.06 (stat.) +/- 0.26 (sys.) +/- 0.29 (cocktail). The enhancement factor exhibits weak centrality and pT dependence in STAR's accessible kinematic regions, while the excess yield in this invariant-mass region as a function of the number of participating nucleons follows a power-law shape with a power of 1.44 +/- 0.10. Models that assume an in-medium broadening of the rho-meson spectral function consistently describe the observed excess in these measurements. Additionally, we report on measurements of omega-and phi-meson production through their e+ e(-) decay channel. These measurements show good agreement with Tsallis blast-wave model predictions, as well as, in the case of the phi meson, results through its K+ K- decay channel. In the intermediate invariant-mass region (1.1 < Mee < 3 GeV / c(2)), we investigate the spectral shapes from different collision centralities. Physics implications for possible in-medium modification of charmed hadron production and other physics sources are discussed.

Autophagy is a multistep process in which cytoplasmic components, including invading pathogens, are captured by autophagosomes that subsequently fuse with degradative lysosomes. Negative-strand RNA viruses, including paramyxoviruses, have been shown to alter autophagy, but the molecular mechanisms remain largely unknown. We demonstrate that human parainfluenza virus type 3 (HPIV3) induces incomplete autophagy by blocking autophagosome-lysosome fusion, resulting in increased virus production. The viral phosphoprotein (P) is necessary and sufficient to inhibition autophagosome degradation. P binds to SNAP29 and inhibits its interaction with syntaxin17, thereby preventing these two host SNARE proteins from mediating autophagosome-lysome fusion. Incomplete autophagy and resultant autophagosome accumulation increase extracellular viral production but do not affect viral protein synthesis. These findings highlight how viruses can block autophagosome degradation by disrupting the function of SNARE proteins.

We demonstrate interface energy level engineering, exploiting the modification in energy band structure across Verwey phase transition of $Fe_{3}O_{4}$ electrode, in a $Fe_{3}O_{4}$(111)/$Alq_{3}$/Co vertical spin-valve (SV) device. Experimental results on device characteristics I-V) study exhibit a transition in conduction mode from carrier injection to tunneling across Verwey transition temperature ($T_{V}$) of $Fe_{3}O_{4}$ electrode. Both giant magneto-resistance (GMR) and tunneling MR (TMR) have been observed in a single SV device as a function of temperature, below and above $T_{V}$, respectively. Appearance of GMR, accompanied by injection limited natural Schottky-like I-V characteristics, provide evidences of spin injection at electrode/$Alq_{3}$ interface and transport through molecular orbitals in this SV device. Features of TMR exhibit significant differences from that of GMR. This is due to the dominant hyperfine-field interaction in the multi-step tunneling regime. We have achieved room-temperature SV operation in our device. A phenomenological model for device operation has been proposed to explain the transition in the conduction mode and associated MR features across $T_{V}$. We propose that the tuning of charge gap at Fermi level across Verwey transition due to charge ordering on the octahedral iron sites of $Fe_{3}O_{4}$ results in a corresponding tuning of conduction mode causing this unique cross over from GMR to TMR in this ferrite-based organic SV.

We report a high precision measurement of the transverse single spin asymmetry A(N) at the center of mass energy root s = 200 GeV in elastic proton-proton scattering by the STAR experiment at RHIC. The A(N) was measured in the four-momentum transfer squared t range 0.003 <= vertical bar t vertical bar <= 0.035 (GeV/c)(2), the region of a significant interference between the electromagnetic and hadronic scattering amplitudes. The measured values of A(N) and its t-dependence are consistent with a vanishing hadronic spin-flip amplitude, thus providing strong constraints on the ratio of the single spin-flip to the non-flip amplitudes. Since the hadronic amplitude is dominated by the Pomeron amplitude at this root s, we conclude that this measurement addresses the question about the presence of a hadronic spin flip due to the Pomeron exchange in polarized proton-proton elastic scattering. (c) 2013 Elsevier B.V. All rights reserved.

ABSTRACT The phosphoprotein (P) of vesicular stomatitis virus (VSV) plays essential roles in viral RNA synthesis. It associates with nascent nucleoprotein (N) to form N 0 -P (free of RNAs), thereby preventing the N from binding to cellular RNAs and maintaining the N in a viral genomic RNA encapsidation-competent form for transcription and replication. The contributions of phosphorylation of P to transcription and replication have been studied intensively, but a concrete mechanism of action still remains unclear. In this study, using a VSV minigenome system, we demonstrated that a mutant of P lacking N-terminal phosphorylation (P3A), in which the N-terminal phosphate acceptor sites are replaced with alanines (S60/A, T62/A, and S64/A), does not support transcription and replication. However, results from protein interaction assays showed that P3A self-associates and interacts with N and the large protein (L) as efficiently as P does. Furthermore, purified recombinant P3A from Sf21 cells supported transcription in an in vitro transcription reconstitution assay. We also proved that P3A is not distributed intranuclearly in vivo . CsCl gradient centrifugation showed that P3A is incapable of preventing N from binding to cellular RNAs and therefore prevents functional template formation. Taken together, our results demonstrate that N-terminal phosphorylation is indispensable for P to prevent N from binding to nonviral RNAs and to maintain the N-specific encapsidation of viral genomic RNA for functional template formation.

On June 4, 2012, the scientific community lost Aaron J. Shatkin, a brilliant scientist, an outstanding mentor, and a truly compassionate human being who touched the lives of his numerous friends and colleagues around the world, including myself. He gallantly fought cancer with his characteristic scientific curiosity and insight, but accepted the ultimate with grace while meticulously carrying out his administrative and scientific investigations, making and receiving phone calls right up to the end. Aaron remains one of a rare breed of scientists who never engaged himself to “do” science but rather to enjoy the excitement of unraveling the complexities of scientific processes. I am extremely fortunate to have joined him as a member of his laboratory in 1969, subsequently remaining as a colleague and collaborator for the next 16 years at the Roche Institute of Molecular Biology (RIMB) in Nutley, New Jersey. I had the privilege of witnessing the inner workings of his mind, his approach to solving scientific problems, and more importantly his way of mentoring associates with enthusiasm and compassion. It was during this time, in 1975, that Aaron made his landmark discovery of a unique cap structure in the 5′-end of reovirus mRNAs, which has had a profound impact on our understanding of the biogenesis of mRNA. Aaron J. Shatkin. Aaron received his undergraduate degree summa cum laude with a major in chemistry in 1956 from Bowdoin College, a renowned liberal arts college in Maine. Years later in 1979, he was recognized by Bowdoin as one of 10 illustrious alumni and Bowdoin conferred on him the Doctor of Science degree. He received his PhD in 1961 under the mentorship of the …

Structural and magnetic properties of 2 at% Mn doped ZnO samples with 48 and 96 hours of milling time were investigated. Structural analysis indicates typical wurtzite structure with greater Mn incorporation in case of 96 hours milled sample. The field dependent magnetization (M-H) measurements confirm ferromagnetic (FM) nature of both the samples. The tendency of variation of saturation magnetization (M-S) with temperature for both samples is expected; however remanent magnetization (M-R) and coercivity (HC) follows an interesting trend with temperature change. The comparatively higher values of M-S, M-R and HC in case of 96 hours milled sample throughout the temperature range from 5K to 300K has been correlated with greater substitutional Mn incorporation in the host ZnO lattice.

Parainfluenza viruses (PIVs) include several members of the Paramyxoviridae family of the Mononegavirales order ranging from Respirovirus, Avulavirus and Rubulavirus genera. These group of viruses consist of several species that causes upper and lower respiratory illness in humans, especially among children and in a variety of animals. These medium-sized, pleomorphic, enveloped viruses attach to the host cells' membranes and get internalized at a neutral pH with the help of haemagglutinin-nuraminidase (HN) and fusion (F) - the two surface glycoproteins. Inside the cytoplasm, the similar to 15 kb of non-segmented negative-sense RNA (NNS) genomes that are encapsidated by the viral nucleocapsid protein (N) are transcribed and replicated by the associated viral polymerase heterocomplex - the large protein (L) and the phosphoprotein (P). The newly replicated RNA genomes are concomitantly encapsidated by the N protein and with the help of the matrix proteins (M), assemble and bud out as mature virions containing the viral glycoproteins and the polymerase along with the host cell membrane envelope. The P gene of PIVs characteristically encodes several accessory proteins, viz C, V, W, D, which are expressed by mRNA editing or alternative translation mechanisms. Despite significant ongoing efforts, no suitable vaccine or antiviral compounds are available to date against these clinically and economically important viruses.

mRNAs of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative strand (NNS) RNA viruses (e.g., rabies, measles, mumps, Ebola, and Borna disease viruses), possess the 5′-terminal cap structure identical to that of eukaryotic mRNAs, but the mechanism of mRNA cap formation is distinctly different from the latter. The elucidation of the unconventional capping of VSV mRNA remained elusive for three decades since the discovery of the cap structure in some viral and eukaryotic mRNAs in 1975. Only recently our biochemical studies revealed an unexpected strategy employed by vesiculoviruses (VSV and Chandipura virus, an emerging arbovirus) to generate the cap structure. This article summarizes the historical and current research that led to the discovery of the novel vesiculoviral mRNA capping reaction.

The RNA-dependent RNA polymerase L protein of vesicular stomatitis virus, a prototype of nonsegmented negative-strand (NNS) RNA viruses, forms a covalent complex with a 5′-phosphorylated viral mRNA-start sequence (L-pRNA), a putative intermediate in the unconventional mRNA capping reaction catalyzed by the RNA:GDP polyribonucleotidyltransferase (PRNTase) activity. Here, we directly demonstrate that the purified L-pRNA complex transfers pRNA to GDP to produce the capped RNA (Gpp-pRNA), indicating that the complex is a bona fide intermediate in the RNA transfer reaction. To locate the active site of the PRNTase domain in the L protein, the covalent RNA attachment site was mapped. We found that the 5′-monophosphate end of the RNA is linked to the histidine residue at position 1,227 (H1227) of the L protein through a phosphoamide bond. Interestingly, H1227 is part of the histidine-arginine (HR) motif, which is conserved within the L proteins of the NNS RNA viruses including rabies, measles, Ebola, and Borna disease viruses. Mutagenesis analyses revealed that the HR motif is required for the PRNTase activity at the step of the enzyme-pRNA intermediate formation. Thus, our findings suggest that an ancient NNS RNA viral polymerase has acquired the PRNTase domain independently of the eukaryotic mRNA capping enzyme during evolution and PRNTase becomes a rational target for designing antiviral agents.

To gain insight into the structural and functional properties of the vesicular stomatitis virus nucleocapsid–RNA complex (vN–RNA), we analyzed it by treatment with proteolytic enzymes. Chymotrypsin treatment to the vN–RNA results in complete digestion of the C-terminal 86 amino acids of the N protein. The residual chymotrypsin resistant vN–RNA complex (vΔN–RNA) carrying N-terminal 336 amino acids of the N protein (ΔN) was inactive in transcription. The ΔN protein retained its capability to protect the genomic RNA from nuclease digestion but failed to interact to the P protein. Interestingly, addition of excess amount of P protein rendered the vN–RNA complex resistant to the chymotrypsin digestion. Finally, our data revealed that the recombinant N–RNA complex purified from bacteria (bN–RNA) is resistant to chymotrypsin digestion, suggesting that the C-terminal unstructured domain (C-loop) remains inaccessible to protease digestion. Detailed comparative analyses of the vN–RNA and vΔN–RNA are discussed.

Human parainfluenza virus type 3 (HPIV3), one of the paramyxoviruses, uses its accessory C protein as an antagonist against interferon (IFN)-mediated host innate immunity. We have previously shown that the C protein significantly decreased the IFN-induced phosphorylation of signal transducer and activator of transcription (Stat) 1 and the formation of gamma IFN activation factor (GAF) complex, thus abrogating the antiviral activity of the IFNs against vesicular stomatitis virus (VSV) replication. Here, by mutational analyses we demonstrated that the N-terminal truncation of the C protein (CNdelta25 and CNdelta50) substantially (approximately 50%) recovers the IFN-induced responses, suggesting the critical role of the N-terminal region of the C protein in IFN signaling. Furthermore, our results indicate that the charged amino acid residues within the N-terminal region of the C protein regulate the antagonistic effect of the C protein on IFN signaling.

Chandipura virus (CHPV) is an emerging human pathogen associated with acute encephalitis and is related closely to vesicular stomatitis virus (VSV), a prototype rhabdovirus. Here, we demonstrate that the RNA polymerase L protein of CHPV exhibits a VSV-like RNA:GDP polyribonucleotidyltransferase (PRNTase) activity, which transfers the 5'-monophosphorylated (p-) viral mRNA start sequence to GDP to produce a capped RNA, and that the conserved HR motif in the CHPV L protein is essential for the PRNTase activity. Interestingly, the CHPV L protein was found to form two distinct SDS-resistant complexes with the CHPV mRNA and leader RNA start sequences; mutations in the HR motif significantly reduced the formation of the former complex (a putative covalent enzyme-pRNA intermediate in the PRNTase reaction), but not the latter complex. These results suggest that the rhabdoviral L proteins universally use the active-site HR motif for the PRNTase reaction at the step of the enzyme-pRNA intermediate formation.

The RNA-dependent RNA-polymerase (RdRp) of human parainfluenza virus type 3 (HPIV3) is a large protein (L, 2233 amino acids), and along with the phosphoprotein (P, 603 amino acids) forms a heterocomplex that transcribes the genome RNA into mRNAs in vitro and in vivo that are 5′-capped and methylated and 3′-polyadenylated. The interaction of the P protein, an obligatory cofactor, imparts the RdRp activity of the L protein, which is otherwise inactive. The precise mechanism underlying this activation process remains unknown. Several recent reports suggested that the L proteins of paramyxoviruses, when expressed alone, self-associate to form an oligomeric structure. The presumptive oligomerization domain lies in the N-terminal part of the L protein (for HPIV3, 889 amino acids). Here, we demonstrate that a series of N-terminally deleted L proteins as well as several truncated proteins that span different regions of the L protein can also efficiently co-immunoprecipitate the full length L protein. In addition, by several biochemical parameters, the L–L interaction was shown to form aggregates rather than oligomers. In contrast, when the P protein was co-expressed with the L protein, the former bound to a domain spanning the N-terminal 1060 amino acids of the latter, which prevented L–L self-association, resulting in the formation of structurally competent and functionally active RdRp.