[show abstract][hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 01/2014; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nu-cleocapsid (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 seques-tered, 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 tetra-mers 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.
Nucleic Acids Research 04/2013; · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Virology 05/2012; 86(15):7777-89. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Virology 07/2011; 85(19):10300-9. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.