Sialic Acid Binding Sites in VP2 of Bluetongue Virus and Their Use during Virus Entry.

Bluetongue virus (BTV), a member of the Orbivirus genus, is transmitted by biting midges (gnats, Culicoides sp.) and is one of the most widespread animal pathogens, causing serious outbreaks in domestic animals, particularly in sheep, with high economic impact. The non-enveloped BTV particle is a double-capsid structure of seven proteins and a genome of 10 double-stranded RNA segments. Although the outermost spike-like VP2 acts as the attachment protein during BTV entry, no specific host receptor has been identified for BTV. Recent high-resolution cryo-electron (cryoEM) structures and biological data have suggested that VP2 may interact with sialic acids (SAs). To confirm this, we have generated protein-based nanoparticles displaying multivalent VP2 and used them to probe glycan arrays. The data show that VP2 binds α2,3-linked SA with high affinity but also binds α2,6-linked SA. Further, Maackia amurensis lectin II (MAL II) and Sambucus nigra lectin (SNA), which specifically bind α2,3-linked and α2,6-linked SAs, respectively, inhibited BTV infection and virus growth in susceptible sheep cells while SNA alone inhibited virus growth in Culicoides-derived cells. A combination of hydrogen deuterium exchange mass spectrometry and site-directed mutagenesis allowed the identification of the specific SA binding residues of VP2. This study provides direct evidence that sialic acids act as key receptor for BTV and that the outer capsid protein VP2 specifically binds SA during BTV entry in both mammalian and insect cells. IMPORTANCE To date no receptor has been assigned for non-enveloped bluetongue virus. To determine if the outermost spike-like VP2 protein is responsible for host cell attachment via interaction with sialic acids, we first generated a protein-based VP2-nanoparticle, for the multivalent presentation of recombinant VP2 protein. Using nanoparticles displaying VP2 to probe a glycan array, we identified that VP2 binds both α2,3-linked and α2,6-linked sialic acids. Lectin inhibitors targeting both linkages of sialic acids showed strong inhibition to BTV infection and progeny virus production in mammalian cells; however the inhibition was only seen with the lectin targeting α2,6-linked sialic acid in insect vector cells. In addition, we identified the VP2 sialic acid binding sites in the exposed tip domain. Our data provides direct evidence that sialic acids act as key receptors for BTV attachment and entry in to both mammalian and insect cells.

Mapping the pH Sensors Critical for Host Cell Entry by a Complex Nonenveloped Virus.

Bluetongue virus (BTV), in the family Reoviridae, is an insect-borne, double-capsid virus causing hemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and various in vitro and in vivo assays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.IMPORTANCE Virus entry into a susceptible cell is the first step of infection and a significant point at which infection can be prevented. To enter effectively, viruses must sense the cellular environment and, when appropriate, initiate a series of changes that eventually jettison the protective shell and deposit virus genes into the cytoplasm. Many viruses sense pH, but how this happens and the events that follow are often poorly understood. Here, we address this question for a large multilayered bluetongue virus. We show key residues in outer capsid proteins, a pH-sensing histidine of a zinc finger within the receptor-binding VP2 protein, and certain histidine residues in the membrane-penetrating VP5 protein that detect cellular pH, leading to irreversible changes and propel the virus through the cell membrane. Our data reveal a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms which may also be used by other viruses.

MiRNA-139-3p inhibits the proliferation, invasion, and migration of human glioma cells by targeting MDA-9/syntenin.

Gliomas are the most common primary malignant brain tumor in adults. Although these tumors are aggressive and frequently lethal, there are currently few therapeutic approaches available to prolong patient survival. MicroRNAs play important roles in regulating the expression of genes that control diverse cellular processes. Here, we investigated the expression and function of miR-139-3p in gliomas using clinical specimens, cultured cells, and a mouse xenograft tumor model. We found that miR-139-3p expression is markedly lower in human glioma tissues than in normal brain tissues. We identified melanoma differentiation-associated gene-9 (MDA-9)/syntenin, an adaptor protein implicated in tumor metastasis, as a novel direct target of miR-139-3p and showed that syntenin mRNA and miR-139-3p levels were inversely correlated in clinical specimens (r = -0.6817, P = 0.0002). Overexpression of miR-139-3p in human glioma cell lines inhibited cell proliferation, migration, and invasion, and these effects were rescued by co-transfection with syntenin. Our results indicate that miR-139-3p plays a significant role in controlling behaviors associated with the malignant progression of gliomas, and we identify the miR-139-3p-syntenin axis as a potential therapeutic target for glioma.

Suprathreshold fiber cluster statistics: Leveraging white matter geometry to enhance tractography statistical analysis.

This work presents a suprathreshold fiber cluster (STFC) method that leverages the whole brain fiber geometry to enhance statistical group difference analyses. The proposed method consists of 1) a well-established study-specific data-driven tractography parcellation to obtain white matter tract parcels and 2) a newly proposed nonparametric, permutation-test-based STFC method to identify significant differences between study populations. The basic idea of our method is that a white matter parcel's neighborhood (nearby parcels with similar white matter anatomy) can support the parcel's statistical significance when correcting for multiple comparisons. We propose an adaptive parcel neighborhood strategy to allow suprathreshold fiber cluster formation that is robust to anatomically varying inter-parcel distances. The method is demonstrated by application to a multi-shell diffusion MRI dataset from 59 individuals, including 30 attention deficit hyperactivity disorder patients and 29 healthy controls. Evaluations are conducted using both synthetic and in-vivo data. The results indicate that the STFC method gives greater sensitivity in finding group differences in white matter tract parcels compared to several traditional multiple comparison correction methods.

Investigating the Influence of Ribavirin on Human Respiratory Syncytial Virus RNA Synthesis by Using a High-Resolution Transcriptome Sequencing Approach.

UNLABELLED: Human respiratory syncytial virus (HRSV) is a major cause of serious respiratory tract infection. Treatment options include administration of ribavirin, a purine analog, although the mechanism of its anti-HRSV activity is unknown. We used transcriptome sequencing (RNA-seq) to investigate the genome mutation frequency and viral mRNA accumulation in HRSV-infected cells that were left untreated or treated with ribavirin. In the absence of ribavirin, HRSV-specific transcripts accounted for up to one-third of total RNA reads from the infected-cell RNA population. Ribavirin treatment resulted in a >90% reduction in abundance of viral mRNA reads, while at the same time no such reduction was detected for the abundance of cellular transcripts. The presented data reveal that ribavirin significantly increases the frequency of HRSV-specific RNA mutations, suggesting a direct influence on the fidelity of the HRSV polymerase. The presented data show that transitions and transversions occur during HRSV replication and that these changes occur in hot spots along the HRSV genome. Examination of nucleotide substitution rates in the viral genome indicated an increase in the frequency of transition but not transversion mutations in the presence of ribavirin. In addition, our data indicate that in the continuous cell types used and at the time points analyzed, the abundances of some HRSV mRNAs do not reflect the order in which the mRNAs are transcribed. IMPORTANCE: Human respiratory syncytial virus (HRSV) is a major pediatric pathogen. Ribavirin can be used in children who are extremely ill to reduce the amount of virus and to lower the burden of disease. Ribavirin is used as an experimental therapy with other viruses. The mechanism of action of ribavirin against HRSV is not well understood, although it is thought to increase the mutation rate of the viral polymerase during replication. To investigate this hypothesis, we used a high-resolution approach that allowed us to determine the genetic sequence of the virus to a great depth of coverage. We found that ribavirin did not cause a detectable change in the relative amounts of viral mRNA transcripts. However, we found that ribavirin treatment did indeed cause an increase in the number of mutations, which was associated with a decrease in virus production.

MicroRNA-105 inhibits human glioma cell malignancy by directly targeting SUZ12.

Glioma accounts for the majority of primary malignant brain tumors in adults and is highly aggressive. Although various therapeutic approaches have been applied, outcomes of glioma treatment remain poor. MicroRNAs are a class of small noncoding RNAs that function as regulators of gene expression. Accumulating evidence shows that microRNAs are associated with tumorigenesis and tumor progression. In this study, we found that miR-105 is significantly downregulated in glioma tissues and glioma cell lines. We identified suppressor of Zeste 12 homolog as a novel direct target of miR-105 and showed that suppressor of Zeste 12 homolog protein levels were inversely correlated with the levels of miR-105 expression in clinical specimens. Overexpression of miR-105 inhibited cell proliferation, tumorigenesis, migration, invasion, and drug sensitivity, whereas overexpression of suppressor of Zeste 12 homolog antagonized the tumor-suppressive functions of miR-105. Taken together, our results indicate that miR-105 plays a significant role in tumor behavior and malignant progression, which may provide a novel therapeutic strategy for the treatment of glioma and other cancers.

MiR-198 enhances temozolomide sensitivity in glioblastoma by targeting MGMT.

Glioblastoma is one of the most frequent and aggressive brain tumors. Accumulating evidence indicates that microRNAs are involved in glioma proliferation, invasion and drug resistance. Previous studies showed that miR-198 is downregulated in glioblastoma. However, the function of miR-198 in glioblastoma is still unclear. In this study, we report that miR-198 levels were greatly downregulated in glioblastoma specimens and decreased expression of miR-198 was associated with poor prognosis in patients with glioblastoma. And overexpression of miR-198 increased chemosensitivity to temozolomide in vitro and in vivo. O6-methylguanine-DNA methyltransferase (MGMT) was identified as a direct target of miR-198, and miR-198 overexpression prevented the protein translation of MGMT. Furthermore, overexpression of MGMT restored miR-198-induced chemosensitivity to temozolomide. Moreover, the protein levels of MGMT were upregulated in clinical glioblastoma specimens and inversely correlated with miR-198 levels. In conclusion, our studies revealed that MiR-198 induces chemosensitivity in glioblastoma by targeting MGMT and that miR-198 may be used as a new diagnostic marker and therapeutic target for glioblastoma in the future.

Crystal structure of the essential transcription antiterminator M2-1 protein of human respiratory syncytial virus and implications of its phosphorylation.

The M2-1 protein of the important pathogen human respiratory syncytial virus is a zinc-binding transcription antiterminator that is essential for viral gene expression. We present the crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.5 Å. The structure reveals that M2-1 forms a disk-like assembly with tetramerization driven by a long helix forming a four-helix bundle at its center, further stabilized by contact between the zinc-binding domain and adjacent protomers. The tetramerization helix is linked to a core domain responsible for RNA binding activity by a flexible region on which lie two functionally critical serine residues that are phosphorylated during infection. The crystal structure of a phosphomimetic M2-1 variant revealed altered charge density surrounding this flexible region although its position was unaffected. Structure-guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data we present here identify surfaces critical for M2-1 function that may be targeted by antiviral compounds.

Elucidation of the Cellular Interactome of Ebola Virus Nucleoprotein and Identification of Therapeutic Targets.

Ebola virus (EBOV) infection results in severe disease and in some cases lethal hemorrhagic fever. The infection is directed by seven viral genes that encode nine viral proteins. By definition, viruses are obligate intracellular parasites and require aspects of host cell biology in order to replicate their genetic material, assemble new virus particles, and subvert host cell antiviral responses. Currently licensed antivirals are targeted against viral proteins to inhibit their function. However, experience with treating HIV and influenza virus demonstrates that resistant viruses are soon selected. An emerging area in virology is to transiently target host cell proteins that play critical proviral roles in virus biology, especially for acute infections. This has the advantage that the protein being targeted is evolutionary removed from the genome of the virus. Proteomics can aid in discovery biology and identify cellular proteins that may be utilized by the virus to facilitate infection. This work focused on defining the interactome of the EBOV nucleoprotein and identified that cellular chaperones, including HSP70, associate with this protein to promote stability. Utilization of a mini-genome replication system based on a recent Makona isolate demonstrated that disrupting the stability of NP had an adverse effect on viral RNA synthesis.

Characterization of the Interactome of the Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 2 Reveals the Hyper Variable Region as a Binding Platform for Association with 14-3-3 Proteins.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a major threat to the swine industry worldwide and hence global food security, exacerbated by a newly emerged highly pathogenic (HP-PRRSV) strain from China. PRRSV nonstructural protein 2 (nsp2) is a multifunctional polypeptide with strain-dependent influences on pathogenicity. A number of discrete functional regions have been identified on the protein. Quantitative label free proteomics was used to identify cellular binding partners of nsp2 expressed by HP-PRRSV. This allowed the identification of potential cellular interacting partners and the discrimination of nonspecific interactions. The interactome data were further investigated and validated using biological replicates and also compared with nsp2 from a low pathogenic (LP) strain of PRRSV. Validation included both forward and reverse pulldowns and confocal microscopy. The data indicated that nsp2 interacted with a number of cellular proteins including 14-3-3, CD2AP, and other components of cellular aggresomes. The hyper-variable region of nsp2 protein was identified as a binding platform for association with 14-3-3 proteins.

Interactome analysis of the human respiratory syncytial virus RNA polymerase complex identifies protein chaperones as important cofactors that promote L-protein stability and RNA synthesis.

UNLABELLED: The human respiratory syncytial virus (HRSV) core viral RNA polymerase comprises the large polymerase protein (L) and its cofactor, the phosphoprotein (P), which associate with the viral ribonucleoprotein complex to replicate the genome and, together with the M2-1 protein, transcribe viral mRNAs. While cellular proteins have long been proposed to be involved in the synthesis of HRSV RNA by associating with the polymerase complex, their characterization has been hindered by the difficulty of purifying the viral polymerase from mammalian cell culture. In this study, enhanced green fluorescent protein (EGFP)-tagged L- and P-protein expression was coupled with high-affinity anti-GFP antibody-based immunoprecipitation and quantitative proteomics to identify cellular proteins that interacted with either the L- or the P-proteins when expressed as part of a biologically active viral RNP. Several core groups of cellular proteins were identified that interacted with each viral protein including, in both cases, protein chaperones. Ablation of chaperone activity by using small-molecule inhibitors confirmed previously reported studies which suggested that this class of proteins acted as positive viral factors. Inhibition of HSP90 chaperone function in the current study showed that HSP90 is critical for L-protein function and stability, whether in the presence or absence of the P-protein. Inhibition studies suggested that HSP70 also disrupts virus biology and might help the polymerase remodel the nucleocapsid to allow RNA synthesis to occur efficiently. This indicated a proviral role for protein chaperones in HRSV replication and demonstrates that the function of cellular proteins can be targeted as potential therapeutics to disrupt virus replication. IMPORTANCE: Human respiratory syncytial virus (HRSV) represents a major health care and economic burden, being the main cause of severe respiratory infections in infants worldwide. No vaccine or effective therapy is available. This study focused on identifying those cellular proteins that potentially interact specifically with the viral proteins that are central to virus replication and transcription, with a view to providing potential targets for the development of a specific, transient therapeutic which disrupts virus biology but prevents the emergence of resistance, while maintaining cell viability. In particular, protein chaperones (heat shock proteins 70 and 90), which aid protein folding and function, were identified. The mechanism by which these chaperones contribute to virus biology was tested, and this study demonstrates to the field that cellular protein chaperones may be required for maintaining the correct folding and therefore functionality of specific proteins within the virus replication complex.

Human respiratory syncytial virus non-structural protein NS1 modifies miR-24 expression via transforming growth factor-ß.

Human respiratory syncytial virus (RSV) is a major health challenge in the young and elderly owing to the lack of a safe and effective vaccine and proven antiviral drugs. Understanding the mechanisms by which viral genes and proteins modulate the host response to infection is critical for identifying novel disease intervention strategies. In this study, the RSV non-structural protein NS1 was shown to suppress miR-24 expression during infection. Lack of NS1 was linked to increased expression of miR-24, whilst NS1 overexpression suppressed miR-24 expression. NS1 was found to induce Kruppel-like factor 6 (KLF6), a transcription factor that positively regulates the transforming growth factor (TGF)-b pathway to induce cell cycle arrest. Silencing of KLF6 led to increased miR-24 expression via downregulation of TGF-ß. Treatment with exogenous TGF-ß suppressed miR-24 expression and induced KLF6. Confocal microscopy showed co-localization of KLF6 and RSV NS1. These findings indicated that RSV NS1 interacts with KLF6 and modulates miR-24 expression and TGF-ß, which facilitates RSV replication.

Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization.

All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N-RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.

Elucidation of the Ebola virus VP24 cellular interactome and disruption of virus biology through targeted inhibition of host-cell protein function.

Viral pathogenesis in the infected cell is a balance between antiviral responses and subversion of host-cell processes. Many viral proteins specifically interact with host-cell proteins to promote virus biology. Understanding these interactions can lead to knowledge gains about infection and provide potential targets for antiviral therapy. One such virus is Ebola, which has profound consequences for human health and causes viral hemorrhagic fever where case fatality rates can approach 90%. The Ebola virus VP24 protein plays a critical role in the evasion of the host immune response and is likely to interact with multiple cellular proteins. To map these interactions and better understand the potential functions of VP24, label-free quantitative proteomics was used to identify cellular proteins that had a high probability of forming the VP24 cellular interactome. Several known interactions were confirmed, thus placing confidence in the technique, but new interactions were also discovered including one with ATP1A1, which is involved in osmoregulation and cell signaling. Disrupting the activity of ATP1A1 in Ebola-virus-infected cells with a small molecule inhibitor resulted in a decrease in progeny virus, thus illustrating how quantitative proteomics can be used to identify potential therapeutic targets.

The interactome of the human respiratory syncytial virus NS1 protein highlights multiple effects on host cell biology.

Viral proteins can have multiple effects on host cell biology. Human respiratory syncytial virus (HRSV) nonstructural protein 1 (NS1) is a good example of this. During the virus life cycle, NS1 can act as an antagonist of host type I and III interferon production and signaling, inhibit apoptosis, suppress dendritic cell maturation, control protein stability, and regulate transcription of host cell mRNAs, among other functions. It is likely that NS1 performs these different roles through interactions with multiple host cell proteins. To investigate this and identify cellular proteins that could interact with NS1, we used quantitative proteomics in combination with green fluorescent protein (GFP)-trap immunoprecipitation and bioinformatic analysis. This analysis identified 221 proteins that were potentially part of complexes that could interact with NS1, with many of these associated with transcriptional regulation as part of the mediator complex, cell cycle regulation, and other functions previously assigned to NS1. Specific immunoprecipitation using the GFP trap was used to confirm the ability of selected cellular proteins to interact individually with NS1. Infection of A549 cells with recombinant viruses deficient in the expression of NS1 and overexpression analysis both demonstrated that NS1 was necessary and sufficient for the enrichment of cells in the G(1) phase of the cell cycle.

Occupational survey-based evidence of health status and welfare problems of workers with pneumoconiosis in China.

Background: Pneumoconiosis is the most dangerous occupational disease in China. According to unofficial records, nearly million migrant workers were affected by pneumoconiosis in 2011, with the number increasing annually. Among them, a large number of migrant workers suffering from pneumoconiosis were not medically diagnosed. Therefore, fundamental questions remain unanswered: what is the background of workers who receive a diagnosis of pneumoconiosis, and how does pneumoconiosis affect their future and well-being? Methods: In this study, we identified and surveyed 1,134 workers with pneumoconiosis in seven selected regions in China with substantially high incidences of pneumoconiosis by using a combination of cluster sampling, convenience sampling, and snowball sampling. We used demographic, medical, and rehabilitation conditions and welfare questionnaires to collect the data. Results: The findings highlighted the socioeconomic status of patients with pneumoconiosis. The majority of workers with pneumoconiosis were adult men who had received no higher education, who lived in rural households, and who were employed in mining or manufacturing industries. Among these workers, 52.8% had been exposed to dust at work for more than 10 years, and 53.1% received a diagnosis of stage II or III pneumoconiosis. More than half of the workers (569 workers, 50.2%) did not receive comprehensive, routine treatment; 33.4% (379 workers) visited a doctor when they experienced physical discomfort, and 6.6% (75 workers) never received treatment. Only 156 workers (13.8%) received rehabilitation services, whereas 978 workers (86.2%) never did. The study results also revealed the severe financial difficulties faced by patients with pneumoconiosis. Only 208 workers (18.3%) had access to work-related injury insurance, with the cost of pneumoconiosis treatment being a substantial burden for 668 workers (60.6%). Conclusion: In this study, we explored the existing health and welfare problems faced by workers with pneumoconiosis in China and identified the social injustice and health disparities that these workers experience. We also clarified the primary challenges in implementing safety, health, and welfare policies for these workers and those who are exposed to high-risk environments, such as those working in mining.

Characterization of the interaction between human respiratory syncytial virus and the cell cycle in continuous cell culture and primary human airway epithelial cells.

Viruses can modify conditions inside cells to make them more favorable for replication and progeny virus production. One way of doing this is through manipulation of the cell cycle, a process that describes the ordered growth and division of cells. Analysis of model cell lines, such as A549 cells and primary airway epithelial cells, infected with human respiratory syncytial virus (HRSV) has shown alteration of the cell cycle during infection, although the signaling events were not clearly understood. In this study, targeted transcriptomic analysis of HRSV-infected primary airway epithelial cells revealed alterations in the abundances of many mRNAs encoding cell cycle-regulatory molecules, including decreases in the D-type cyclins and corresponding cyclin-dependent kinases (CDK4 and CDK6 [CDK4/6]). These alterations were reflected in changes in protein abundance and/or relocalization in HRSV-infected cells; taken together, they were predicted to result in G(0)/G(1) phase arrest. In contrast, there was no change in the abundances of D-type cyclins in A549 cells infected with HRSV. However, the abundance of the G(1)/S phase progression inhibitor p21(WAF1/CIP1) was increased over that in mock-treated cells, and this, again, was predicted to result in G(0)/G(1) phase arrest. The G(0)/G(1) phase arrest in both HRSV-infected primary cells and A549 cells was confirmed using dual-label flow cytometry that accurately measured the different stages of the cell cycle. Comparison of progeny virus production in primary and A549 cells enriched in G(0)/G(1) using a specific CDK4/6 kinase inhibitor with asynchronously replicating cells indicated that this phase of the cell cycle was more efficient for virus production.

Different NF-κB activation characteristics of human respiratory syncytial virus subgroups A and B.

Human respiratory syncytial virus (HRSV) is a member of the family Paramyxoviridae, and is responsible for serious respiratory illness in infants, the elderly and the immunocompromised. HRSV exists as two distinct lineages known as subgroups A and B, which represent two lines of divergent evolution with extensive genetic and serologic differences. While both subgroup A and B viruses contribute to overall HRSV disease, subgroup A isolates are associated with both increased frequency and morbidity of infections, and reasons for this are unclear. HRSV disease is characterized by virus-mediated cell destruction in combination with extensive inflammatory and immune modulatory responses, and for HRSV subgroup A isolates, several of these signaling pathways are regulated through activation of the transcription factor NF-κB. In contrast, the NF-κB activation characteristics of HRSV subgroup B infection remain untested. Here, we performed a quantitative and comparative analysis of NF-κB activation in response to infection of both continuous and primary cell cultures with HRSV subgroup A and B isolates. Our results showed the model HRSV subgroup A isolate consistently induced increased NF-κB activation compared to its HRSV subgroup B counterpart. The differential NF-κB activation characteristics of HRSV subgroup A and B viruses may contribute to differences in their pathogenesis.

Quantitative proteomic analysis of A549 cells infected with human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is a major cause of pediatric lower respiratory tract disease to which there is no vaccine or efficacious chemotherapeutic strategy. Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. Quantitative proteomics was used to take an unbiased overview of the protein changes in transformed human alveolar basal epithelial cells infected with HRSV. Underpinning this was the use of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS, which allowed the direct and simultaneous identification and quantification of both cellular and viral proteins. To reduce sample complexity and increase data return on potential protein localization, cells were fractionated into nuclear and cytoplasmic extracts. This resulted in the identification of 1,140 cellular proteins and six viral proteins. The proteomics data were analyzed using Ingenuity Pathways Analysis to identify defined canonical pathways and functional groupings. Selected data were validated using Western blot, direct and indirect immunofluorescence confocal microscopy, and functional assays. The study served to validate and expand upon known HRSV-host cell interactions, including those associated with the antiviral response and alterations in subnuclear structures such as the nucleolus and ND10 (promyelocytic leukemia bodies). In addition, novel changes were observed in mitochondrial proteins and functions, cell cycle regulatory molecules, nuclear pore complex proteins and nucleocytoplasmic trafficking proteins. These data shed light into how the cell is potentially altered to create conditions more favorable for infection. Additionally, the study highlights the application and advantage of stable isotope labeling with amino acids in cell culture coupled to LC-MS/MS for the analysis of virus-host interactions.

Redescription and phylogenetic position of the terrestrial ciliates Gastrostylides dorsicirratus and Heterourosomoida lanceolata (Hypotricha, Dorsomarginalia).

The morphology and infraciliature of two soil hypotrichous ciliates, Heterourosomoida lanceolata (Shibuya, 1930) Singh and Kamra, 2015 and Gastrostylides dorsicirratus (Foissner, 1982) Foissner, 2016, were investigated using live observation and protargol staining. The Chinese population of H. lanceolata differs slightly from other populations in the body size in vivo, the relative length of the adoral zone, the number of right and left marginal cirri, the total number of dorsal bristles, and the number of micronuclei. The Chinese population of G. dorsicirratus corresponds well with Austrian and Indian populations. We also document the morphogenetic processes during binary fission of G. dorsicirratus, including the formation of the frontoventral-transverse cirral anlagen in a primary pattern. In addition, phylogenetic analyses based on SSU rDNA sequence data reveal that the two populations of G. dorsicirratus for which data are available cluster together with full support and form a clade with Heterourosomoida sinica and two populations of H. lanceolata.