Integration host factor (IHF) is an Escherichia coli protein involved in (i) condensation of the bacterial nucleoid and (ii) regulation of a variety of cellular functions. In its regulatory role, IHF binds to a specific sequence to introduce a strong bend into the DNA; this provides a duplex architecture conducive to the assembly of site-specific nucleoprotein complexes. Alternatively, the protein can bind in a sequence-independent manner that weakly bends and wraps the duplex to promote nucleoid formation. IHF is also required for the development of several viruses, including bacteriophage lambda, where it promotes site-specific assembly of a genome packaging motor required for lytic development. Multiple IHF consensus sequences have been identified within the packaging initiation site (cos), and we here interrogate IHF–cos binding interactions using complementary electrophoretic mobility shift (EMS) and analytical ultracentrifugation (AUC) approaches. IHF recognizes a single consensus sequence within cos (I1) to afford a strongly bent nucleoprotein complex. In contrast, IHF binds weakly but with positive cooperativity to nonspecific DNA to afford an ensemble of complexes with increasing masses and levels of condensation. Global analysis of the EMS and AUC data provides constrained thermodynamic binding constants and nearest neighbor cooperativity factors for binding of IHF to I1 and to nonspecific DNA substrates. At elevated IHF concentrations, the nucleoprotein complexes undergo a transition from a condensed to an extended rodlike conformation; specific binding of IHF to I1 imparts a significant energy barrier to the transition. The results provide insight into how IHF can assemble specific regulatory complexes in the background of extensive nonspecific DNA condensation.
Genome packaging is strongly conserved in the complex double-stranded DNA viruses, including the herpesviruses and many bacteriophages. In these cases, viral DNA is packaged into a procapsid shell by a terminase enzyme. The packaging substrate is typically a concatemer composed of multiple genomes linked in a head-to-tail fashion, and terminase enzymes perform two essential functions: 1) excision of a unit length genome from the concatemer (genome maturation) and 2) translocation of the duplex into a procapsid (genome packaging). While the packaging motors have been described in some detail, the maturation complexes remain ill characterized. Here we describe the assembly, physical characteristics, and catalytic activity of the λ-genome maturation complex. The λ-terminase protomer is composed of one large catalytic subunit tightly associated with two DNA recognition subunits. The isolated protomer binds DNA weakly and does not discriminate between nonspecific DNA and duplexes that contain the packaging initiation sequence, cos. The Escherichia coli integration host factor protein (IHF) is required for efficient λ-development in vivo and a specific IHF recognition sequence is found within cos. We show that IHF and the terminase protomer cooperatively assemble at the cos site and that the small terminase subunit plays the dominant role in complex assembly. Analytical ultracentrifugation analysis reveals that the maturation complex is composed of four protomers and one IHF heterodimer bound at the cos site. Tetramer assembly activates the cos-cleavage nuclease activity of the enzyme, which matures the genome end in preparation for packaging. The stoichiometry and catalytic activity of the complex is reminiscent of the type IIE and IIF restriction endonucleases and the two systems may share mechanistic features. This study, to our knowledge, provides our first detailed glimpse into the structural and functional features of a viral genome maturation complex, an essential intermediate in the development of complex dsDNA viruses.
generic icosahedral capsid assembly or to studies of ranges of possibilities found over broad parameter domains. We describe work intended to help bridge this gap between theoretical models of capsid assembly in general and experimental work on specific model systems by using computational parameter estimation to learn rate parameters for stochastic simulations of capsid assembly from available experimental data. Our method combines ideas from gradient-based and response-surface optimization methods with a heuristic global search strategy to find parameter fits that approximately reproduce experimental measures of overall assembly progress. We demonstrate the approach through application to light scattering data tracking assembly progress of several in vitro capsid assembly systems. The results provide insight into possible mechanisms and pathways of assembly for specific capsid systems in vitro. They further provide a basis for future studies attempting to computationally project how behavior of these systems would be altered in conditions more closely approximating those expected at sites of capsid assembly in vivo. 2180-Pos Board B166Coarse-Grained Molecular Dynamics Simulations of the Entire Influenza Virus Envelope Daniel L. Parton, Marc Baaden, Mark S.P. Sansom. The envelope of the influenza virus contains three membrane proteins: hemagglutinin (HA), neuraminidase (NA) and the M2 proton channel. The interactions of these proteins with their surrounding lipid environment are important for many phases of the viral life cycle. In the various membranes of an infected host cell, newly formed viral proteins are thought to use lipid rafts -small patches of ordered membrane -to locate themselves at the plasma membrane. The arrangement of the proteins within the envelope of free virions may also be important for the infectivity of the virus. We have used the MARTINI coarse-grained force field to simulate a viral envelope of realistic size for several microseconds. Coarse-grained methods allow simulations on large systems (4.5 million particles for the system in this work) over extended timescales. Using information from recent cryoelectron tomography images of complete virions as a basis, our model has been constructed as a 60 nm diameter lipid vesicle with 80 HA, 12 NA and 12 M2 proteins inserted in the membrane. The protein structures are derived from existing crystallographic and NMR structures. The vesicle membrane is a ternary mixture of saturated and poly-unsaturated phospholipids, and cholesterol, which has been shown in other work to separate into raft and non-raft phases. The simulations will be analysed to provide information on the structural and dynamical properties of the viral envelope. In particular, we will focus on the partitioning of proteins between raft and non-raft lipid domains, and the degree of protein clustering. 2181-Pos Board B167Reducing Immune Response against Lentiviral Vectors: Lentiviral Vector Presentation of CD47, The 'Marker of Self' Nisha Sosale, Richard K. Tsai, Irena Ivanovska,...
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