Molecular Dissection of the Forces Responsible for Viral Capsid Assembly and Stabilization by Decoration Proteins.

Complex double-stranded DNA viruses utilize a terminase enzyme to package their genomes into a preassembled procapsid shell. DNA packaging triggers a major conformational change in the proteins assembled into the shell and most often subsequent addition of a decoration protein that is required to stabilize the structure. In bacteriophage λ, DNA packaging drives a procapsid expansion transition to afford a larger but fragile shell.

Thermodynamic Interrogation of the Assembly of a Viral Genome Packaging Motor Complex.

Viral terminase enzymes serve as genome packaging motors in many complex double-stranded DNA viruses. The functional motors are multiprotein complexes that translocate viral DNA into a capsid shell, powered by a packaging ATPase, and are among the most powerful molecular motors in nature. Given their essential role in virus development, the structure and function of these biological motors is of considerable interest.

Assembly and characterization of gp160-nanodiscs: A new platform for biochemical characterization of HIV envelope spikes.

The human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS) and is thus responsible for significant morbidity and mortality worldwide. Despite considerable effort, preparation of an effective vaccine for AIDS has been elusive and it has become clear that a fundamental understanding of the relevant antigenic targets on HIV is essential. The Env trimer spike is the only viral antigen present on the surface of the viral particle and it is the target of all broadly neutralizing antibodies isolated to date.

Analytical Ultracentrifugation as a Tool to Study Nonspecific Protein-DNA Interactions.

Analytical ultracentrifugation (AUC) is a powerful tool that can provide thermodynamic information on associating systems. Here, we discuss how to use the two fundamental AUC applications, sedimentation velocity (SV), and sedimentation equilibrium (SE), to study nonspecific protein-nucleic acid interactions, with a special emphasis on how to analyze the experimental data to extract thermodynamic information. We discuss three specific applications of this approach: (i) determination of nonspecific binding stoichiometry of E.

Integration host factor assembly at the cohesive end site of the bacteriophage lambda genome: implications for viral DNA packaging and bacterial gene regulation.

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.

Major capsid reinforcement by a minor protein in herpesviruses and phage.

Herpes simplex type 1 virus (HSV-1) and bacteriophage λ capsids undergo considerable structural changes during self-assembly and DNA packaging. The initial steps of viral capsid self-assembly require weak, non-covalent interactions between the capsid subunits to ensure free energy minimization and error-free assembly. In the final stages of DNA packaging, however, the internal genome pressure dramatically increases, requiring significant capsid strength to withstand high internal genome pressures of tens of atmospheres.

A pseudo-atomic model for the capsid shell of bacteriophage lambda using chemical cross-linking/mass spectrometry and molecular modeling.

Bacteriophage lambda is one of the most exhaustively studied of the double-stranded DNA viruses. Its assembly pathway is highly conserved among the herpesviruses and many of the bacteriophages, making it an excellent model system. Despite extensive genetic and biophysical characterization of many of the lambda proteins and the assembly pathways in which they are implicated, there is a relative dearth of structural information on many of the most critical proteins involved in lambda assembly and maturation, including that of the lambda major capsid protein.

Strong subunit coordination drives a powerful viral DNA packaging motor.

Terminase enzymes are viral motors that package DNA into a preformed capsid and are of interest both therapeutically and as potential nano-machines. The enzymes excise a single genome from a concatemeric precursor (genome maturation) and then package the duplex to near-crystalline density (genome packaging). The functional motors are oligomers of protomeric subunits and are the most powerful motors currently known.

The enzymology of a viral genome packaging motor is influenced by the assembly state of the motor subunits.

Terminase enzymes are responsible for the excision of a single genome from a concatemeric precursor (genome maturation) and concomitant packaging of DNA into the capsid shell. Here, we demonstrate that lambda terminase can be purified as a homogeneous "protomer" species, and we present a kinetic analysis of the genome maturation and packaging activities of the protomeric enzyme. The protomer assembles into a distinct maturation complex at the cos sequence of a concatemer.

Thermodynamic characterization of viral procapsid expansion into a functional capsid shell.

The assembly of "complex" DNA viruses such as the herpesviruses and many tailed bacteriophages includes a DNA packaging step where the viral genome is inserted into a preformed procapsid shell. Packaging triggers a remarkable capsid expansion transition that results in thinning of the shell and an increase in capsid volume to accept the full-length genome. This transition is considered irreversible; however, here we demonstrate that the phage λ procapsid can be expanded with urea in vitro and that the transition is fully reversible.

Challenging packaging limits and infectivity of phage λ.

The terminase motors of bacteriophages have been shown to be among the strongest active machines in the biomolecular world, being able to package several tens of kilobase pairs of viral genome into a capsid within minutes. Yet, these motors are hindered at the end of the packaging process by the progressive buildup of a force-resisting packaging associated with already packaged DNA. In this experimental work, we raise the issue of what sets the upper limit on the length of the genome that can be packaged by the terminase motor of phage λ and still yield infectious virions and the conditions under which this can be efficiently performed.

The bacteriophage lambda gpNu3 scaffolding protein is an intrinsically disordered and biologically functional procapsid assembly catalyst.

Procapsid assembly is a process whereby hundreds of copies of a major capsid protein assemble into an icosahedral protein shell into which the viral genome is packaged. The essential features of procapsid assembly are conserved in both eukaryotic and prokaryotic complex double-stranded DNA viruses. Typically, a portal protein nucleates the co-polymerization of an internal scaffolding protein and the major capsid protein into an icosahedral capsid shell.

Assembly and maturation of the bacteriophage lambda procapsid: gpC is the viral protease.

Viral capsids are robust structures designed to protect the genome from environmental insults and deliver it to the host cell. The developmental pathway for complex double-stranded DNA viruses is generally conserved in the prokaryotic and eukaryotic groups and includes a genome packaging step where viral DNA is inserted into a pre-formed procapsid shell. The procapsids self-assemble from monomeric precursors to afford a mature icosahedron that contains a single "portal" structure at a unique vertex; the portal serves as the hole through which DNA enters the procapsid during particle assembly and exits during infection.

Packaging of a unit-length viral genome: the role of nucleotides and the gpD decoration protein in stable nucleocapsid assembly in bacteriophage lambda.

The developmental pathways for a variety of eukaryotic and prokaryotic double-stranded DNA viruses include packaging of viral DNA into a preformed procapsid structure, catalyzed by terminase enzymes and fueled by ATP hydrolysis. In most instances, a capsid expansion process accompanies DNA packaging, which significantly increases the volume of the capsid to accommodate the full-length viral genome. "Decoration" proteins add to the surface of the expanded capsid lattice, and the terminase motors tightly package DNA, generating up to approximately 20 atm of internal capsid pressure.

Biophysics of viral infectivity: matching genome length with capsid size.

In this review, we discuss recent advances in biophysical virology, presenting experimental and theoretical studies on the physical properties of viruses. We focus on the double-stranded (ds) DNA bacteriophages as model systems for all of the dsDNA viruses both prokaryotic and eukaryotic. Recent studies demonstrate that the DNA packaged into a viral capsid is highly pressurized, which provides a force for the first step of passive injection of viral DNA into a bacterial cell.

Assembly of bacteriophage lambda terminase into a viral DNA maturation and packaging machine.

Terminase enzymes are common to complex double-stranded DNA viruses and function to package viral DNA into the capsid. We recently demonstrated that the bacteriophage lambda terminase gpA and gpNu1 proteins assemble into a stable heterotrimer with a molar ratio gpA1/gpNu1(2). This terminase protomer possesses DNA maturation and packaging activities that are dependent on the E.

Building a virus from scratch: assembly of an infectious virus using purified components in a rigorously defined biochemical assay system.

The assembly of double-stranded DNA (dsDNA) viruses such as poxvirus, the herpesviruses and many bacteriophages is a complex process that requires the coordinated activities of numerous proteins of both viral and host origin. Here, we report the assembly of an infectious wild-type lambda virus using purified proteins and commercially available DNA, and optimization of the assembly reaction in a rigorously defined biochemical system. Seven proteins, purified procapsids and tails, and mature lambda DNA are necessary and sufficient for efficient virus assembly in vitro.

Self-association properties of the bacteriophage lambda terminase holoenzyme: implications for the DNA packaging motor.

Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of viral DNA into a protective capsid. Bacteriophage lambda terminase holoenzyme is a hetero-oligomer composed of the A and Nu1 lambda gene products; however, the self-association properties of the holoenzyme have not been investigated systematically. Here, we report the results of sedimentation velocity, sedimentation equilibrium, and gel-filtration experiments studying the self-association properties of the holoenzyme.

A minimal kinetic model for a viral DNA packaging machine.

Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes possess ATPase and nuclease activities that work in concert to "package" a viral genome into an empty procapsid, and it is likely that terminase enzymes from disparate viruses utilize a common packaging mechanism. Bacteriophage lambda terminase possesses a site-specific nuclease activity, a so-called helicase activity, a DNA translocase activity, and multiple ATPase catalytic sites that function to package viral DNA.

Biochemical characterization of bacteriophage lambda genome packaging in vitro.

Bacteriophage lambda has been extensively studied, and the abundance of genetic and biochemical information available makes this an ideal model system to study virus DNA packaging at the molecular level. Limited in vitro packaging efficiency has hampered progress toward this end, however. It has been suggested that limited packaging efficiency is related to poor activity of purified procapsids.

Insights into specific DNA recognition during the assembly of a viral genome packaging machine.

Terminase enzymes mediate genome "packaging" during the reproduction of DNA viruses. In lambda, the gpNu1 subunit guides site-specific assembly of terminase onto DNA. The structure of the dimeric DNA binding domain of gpNu1 was solved using nuclear magnetic resonance spectroscopy.