Carlos E. Catalano - US grants

The objective of this proposal is to examine, at the molecular level, the mechanism of assembly of viral precursors into an infectious virus particle. One of the final steps in viral assembly is the packaging of the viral genome into a protective protein coat known as the capsid, or head. Similar mechanisms for DNA packaging have been proposed for all of the double-stranded DNA bacteriophages and may also apply to mammalian viruses such as adenovirus and herpesvirus. Terminases are enzymes common to all of these viruses and are responsible for packaging of a single genome from a concatameric precursor. Bacteriophage lambda has been extensively studied over the years and represents an ideal system in which to study viral DNA packaging. We therefore propose to use phage lambda terminase as a model enzyme with which to study DNA packaging and virus assembly. The present project focuses on the catalytic activities of lambda terminase and their role in the packaging of viral DNA. This enzyme possesses a site-specific endonuclease activity, a DNA-stimulated ATPase activity and a DNA helicase activity, all of which work in concert to effect genome packaging. DNA packaging by lambda terminase initiates with the assembly of a stable multiprotein complex (termasome) onto the concatameric packaging substrate and site-specific nicking of the duplex. Prior to or immediately after strand separation by the enzyme, an empty prohead binds to the binary protein.DNA intermediate and the termasome releases from the assembly site. Translocation of terminase ensues, likely powered by the hydrolysis of ATP, and DNA is actively packaged into the viral prohead. The experiments described in this proposal systematically probe the protein-protein and protein-DNA interactions required to assemble a stable enzyme.DNA intermediate which nicks the DNA duplex, and the subsequent interactions required to disengage the complex so that packaging may ensue. The interplay between the three catalytic activities in the assembly-release processes as well as the role of ATP and ATP hydrolysis in these functions are examined in detail. Genetic studies have identified several mutant enzymes which are deficient in specific aspects of lambda assembly and these altered proteins will be utilized as tools to further probe the catalytic mechanisms of the packaging process. Both biophysical and kinetic techniques are utilized to examine in detail each of the steps involved in the initiation of genome packaging. While the mechanistic details differ, the data derived from these experiments may be used to model DNA packaging by all of the double- stranded DNA phages, and may include assembly in the eucaryotic adenovirus and herpesvirus groups. Mechanistic similarities to other DNA manipulating enzymes such as the recBCD nuclease/helicase of E. coli, the restriction endonucleases, particularly types IIS and III, and the assembly-release of an open-promoter complex during the initiation of transcription further suggest that an understanding of the catalytic properties of this packaging machine may yield insight into the general mechanisms of DNA manipulation by multiprotein enzyme complexes.

DESCRIPTION (provided by the applicant): The assembly of nucleoprotein structures that perform complex biological functions is a common theme in nature. Relevant examples include the nucleoprotein complexes involved in chromosomal replication, DNA transcription, and genome "packaging" in many double-stranded DNA (dsDNA) viruses. Terminase enzymes are common to a diverse group of dsDNA viruses, including the herpesvirus groups. These enzymes are an integral part of a series of nucleoprotein complexes critical to genome packaging. All of the known terminases possess ATPase and nuclease activities that play an important role in the packaging process. The principal investigator and his group are interested in the biochemical properties of the packaging machinery, and specifically the enzymology of the terminase enzymes. Studies described in this proposal seek to understand the molecular mechanism of genome packaging by lambda terminase. The enzyme possesses site-specific nuclease and ATPase catalytic activities that work in concert to package viral DNA. Viable models describing the packaging pathway have been developed. Many aspects of the packaging pathway remain obscure, however, and most lack mechanistic rigor. Studies are designed to provide a mechanistic basis for the delicate interplay between these catalytic activities, and their role in the assembly and stability of nucleoprotein packaging intermediates. Biochemical and enzyme kinetic studies are designed to directly probe the catalytic mechanism of the enzyme, and to define the role of catalysis in nucleoprotein complex assembly and function. While mechanistic details may differ, the data derived from these experiments may be used to model DNA packaging by other double-stranded DNA viruses, including the medically relevant adenovirus and herpesvirus groups. Furthermore, this enzyme shares mechanistic similarities to the complex nucleoprotein machines involved in DNA replication and transcription. An understanding of the catalytic properties of this packaging machine may yield insight into the general mechanisms of DNA manipulation by large multiprotein enzyme complexes.

Our long-range goal is to understand virus assembly at the molecular level. DNA packaging is a critical step in the assembly of many double-stranded DNA viruses, including poxvirus, adenovirus, herpesviruses, and many bacteriophages. Terminase enzymes are common to these viruses and function to package viral DNA into the capsid. Common mechanisms for genome packaging have been proposed for these all viruses. The process of genome packaging is unique to viruses, and thus represents an ideal target for antiviral therapy;however, the lack of mechanistic detail precludes a rational approach to drug design. Bacteriophage lambda presents an ideal system to study DNA packaging. Lambda terminase is a central component of an ordered series of packaging intermediates, and is an integral part of the packaging motor. The goal of this project is to define a coherent physical and kinetic model for the assembly of a viral DNA packaging motor. A mechanistic model for any complex biological process requires a description of the physical nature of the macromolecular complexes involved, a kinetic dissection of the catalytic activities required for the process, and a mechanism that clearly describes the linkage between catalysis, structure, and function. Unfortunately, virtually nothing is known about the physical properties of the packaging intermediates in lambda. Moreover, a mechanistic link between catalytic activity and function remains elusive, due in part to the dearth of structural information. This project seeks to (i) characterize the interactions responsible for specific assembly of the packaging motor on viral DNA, (ii) define the assembly state of the functional packaging motor, to (iii) characterize critical packaging intermediates, and (iv) characterize the activity of defined enzyme species to provide a direct link between stucture and function of termiase. A combined structural, biophysical, and kinetic approach will define critical aspects central to our understanding of the mechanism and enzymology of DNA packaging.

DESCRIPTION (provided by applicant): The Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome, a devastating infection that has reached pandemic proportions. The first and pre- requisite step for HIV entry into a permissive cell is specific binding of the viral glycoprotein gp120 to the cellular CD4 receptor;this interaction triggers an ordered series of steps that ultimately results in fusion of the viral envelope with the cellular membrane and entry of the nucleocapsid into the cell interior. Gp120 is tethered to the viral envelope via non-covalent interactions with trimeric gp41, an integral membrane protein that mediates membrane fusion events. Trimeric gp120/gp41 spikes represent the primary epitope exposed on the viral surface and a variety of trimeric "gp140" deletion constructs and soluble monomeric gp120 constructs have been studied as immunogens to elicit broadly neutralizing antibodies (bNtAbs) to HIV. Despite heroic efforts, induction of bNtAbs against primary isolates of HIV has been uniformly unsuccessful. This has led to the conclusion that a fundamental understanding of the HIV envelope glycoprotein (Env) spike structure and its interaction with bNtAbs is key to future vaccine development. Unfortunately, this goal has been frustrated by the lack of an in vitro system that allows the study of Env trimers in a soluble, biologically relevant, and functional lipid-bound conformation. Phospholipid nanodiscs provide such a system. Nanodiscs are derived from high-density lipoprotein particles involved in reverse cholesterol transport in humans;they provide stable model membranes into which membrane proteins can be embedded in a native and functional form. They are homogenous in size and defined in composition, and provide a stable, soluble, and mono-disperse platform that is amenable to rigorous biochemical, biophysical, and structural interrogation. We propose to utilize nanodisc technology to assemble HIV Env trimers in a defined lipid bilayer and to characterize the structure and function of the trimeric spikes. This application represents a "proof-of-concept" proposal that is ideally suited to an R21 application as it represents modest-risk, high yield proposal. Successful completion of the stated goals will provide a novel platform to study Env structure and function and provide the foundation for future studies that will utilize this innovative immunogen for HIV vaccine development. PUBLIC HEALTH RELEVANCE: HIV infection has reached pandemic proportions and the development of an effective vaccine will be paramount to containing AIDS. Previous attempts at vaccine development have been disappointing, in large part because the "rationally designed" immunogens have failed to mimic the natural presentation of the HIV envelope (Env) spike structure. We propose to utilize nanodisc technology to assemble and characterize Env spikes in a soluble lipid bilayer. This work will provide the foundation for future studies that will utilize this innovative immunogen for HIV vaccine development.

DESCRIPTION (provided by applicant): Biophysical, Biochemical, and Genetic Analysis A key step in the assembly of many viruses, including herpesviruses and poxviruses that cause significant morbidity and mortality in the human population, is the packaging of dsDNA into pre-assembled procapsids by an ATP-driven motor complex. Viral terminases comprise a major class of these packaging motors and carry out multiple functions, including binding and cleavage of DNA to initiate packaging of a genome-length of DNA from a concatemeric substrate, translocation of the DNA into the procapsid, and arrest and DNA cleavage to terminate the packaging reaction. We propose integrated genetic, biochemical, and biophysical studies to elucidate detailed mechanisms of the phage ? terminase packaging motor, a powerful model system for investigating general principles. Genetic methods are designed to identify mutants with altered packaging activities and determine phenotypic defects in vivo. Biochemical and kinetic studies are designed to interrogate packaging kinetics and assembly of viruses in vitro with defined sets of purified proteins. Biophysical analysis using optical tweezers enables detailed measurements of the packaging of single DNA molecules in real time. Each approach is designed to complement and support the others. The studies will focus on: (1) Identification of amino acid residues directly involved in motor function via detailed studies of the effect of mutations on motor subunit assembly, packaging efficiency and kinetics, ATP consumption, and infectious viral assembly;(2) A mechanistic dissection of the translocating motor to define DNA translocation rate, motor force generation, translocation step size and stepping dynamics, and coordination of motor subunits;(3) Interrogation of packaging termination and genome end maturation to define the physiokinetic factors that mediate sensing of the extent of packaging and motor arrest and DNA cleavage. The proposed studies will utilize a diverse scientific toolbox and build on solid preliminary studies that establish the genetic, biochemical, and biophysical framework used to dissect motor function. These studies will provide an unprecedented understanding of mechanochemical coupling (energy transduction) in the viral packaging motor and will yield mechanistic insight into key steps in virus assembly. The results will guide future studies on other virus systems and help to define general principles of ATP-driven molecular motors relevant to understanding homologous cellular complexes including RNA helicases and chromosome segregation factors. PUBLIC HEALTH RELEVANCE: Our research is aimed at understanding viral DNA packaging, a key step in the assembly of many viruses, including herpesviruses and poxviruses that cause significant morbidity and mortality in the human population. Our studies of the basic principles of virus assembly will lead to a better understanding of this complex biological process and aid in the development of novel antiviral therapeutics.

DESCRIPTION (provided by applicant): Biophysical, Biochemical, and Genetic Analysis A key step in the assembly of many viruses, including herpesviruses and poxviruses that cause significant morbidity and mortality in the human population, is the packaging of dsDNA into pre-assembled procapsids by an ATP-driven motor complex. Viral terminases comprise a major class of these packaging motors and carry out multiple functions, including binding and cleavage of DNA to initiate packaging of a genome-length of DNA from a concatemeric substrate, translocation of the DNA into the procapsid, and arrest and DNA cleavage to terminate the packaging reaction. We propose integrated genetic, biochemical, and biophysical studies to elucidate detailed mechanisms of the phage ? terminase packaging motor, a powerful model system for investigating general principles. Genetic methods are designed to identify mutants with altered packaging activities and determine phenotypic defects in vivo. Biochemical and kinetic studies are designed to interrogate packaging kinetics and assembly of viruses in vitro with defined sets of purified proteins. Biophysical analysis using optical tweezers enables detailed measurements of the packaging of single DNA molecules in real time. Each approach is designed to complement and support the others. The studies will focus on: (1) Identification of amino acid residues directly involved in motor function via detailed studies of the effect of mutations on motor subunit assembly, packaging efficiency and kinetics, ATP consumption, and infectious viral assembly; (2) A mechanistic dissection of the translocating motor to define DNA translocation rate, motor force generation, translocation step size and stepping dynamics, and coordination of motor subunits; (3) Interrogation of packaging termination and genome end maturation to define the physiokinetic factors that mediate sensing of the extent of packaging and motor arrest and DNA cleavage. The proposed studies will utilize a diverse scientific toolbox and build on solid preliminary studies that establish the genetic, biochemical, and biophysical framework used to dissect motor function. These studies will provide an unprecedented understanding of mechanochemical coupling (energy transduction) in the viral packaging motor and will yield mechanistic insight into key steps in virus assembly. The results will guide future studies on other virus systems and help to define general principles of ATP-driven molecular motors relevant to understanding homologous cellular complexes including RNA helicases and chromosome segregation factors. PUBLIC HEALTH RELEVANCE: Our research is aimed at understanding viral DNA packaging, a key step in the assembly of many viruses, including herpesviruses and poxviruses that cause significant morbidity and mortality in the human population. Our studies of the basic principles of virus assembly will lead to a better understanding of this complex biological process and aid in the development of novel antiviral therapeutics.

Project Summary. Bacteriophages play a major role in bacterial evolution, in mediating bacterial pathogenicity and antibiotic resistance, in modulating the human microbiome and they have great potential as nanotherapeutics. Understanding these issues with respect to human disease and harnessing their potential as theranostic agents requires a fundamental understanding of virus development. The genome packaging pathways are strongly conserved in the large double-stranded DNA (dsDNA) viruses, both prokaryotic and eukaryotic. In this broad class of viruses, a terminase enzyme is responsible for (i) excision of an individual genome from concatemeric substrate (genome maturation) and (ii) translocation of DNA into a procapsid shell (genome packaging). These functions are catalyzed by terminase enzymes assembled into discrete maturation and packaging motor complexes. Terminases are composed of a catalytic subunit and a DNA recognition subunit, both of which are essential for genome packaging in vivo. Structural and single-molecule studies have provided insight into packaging motor complexes composed of the catalytic subunit in isolation; however, there is little information on motor complexes containing both essential subunits. Further, there is a dearth of structural information on the equally essential maturation complex precursor. This is due, in part, to the absence of well-characterized holoenzyme preparations and a dearth of in vitro assays to comprehensively assess the pathway. We have developed rigorous assays in which the biochemical, biophysical and structural features of the lambda genome-packaging pathway can be defined in great detail. Using these tools, we propose to characterize the structural (cryo-electron microscopy) and functional (biophysical, kinetic) features of the maturation complex, which show mechanistic similarity to the tetrameric type IIE/F restriction endonucleases. We directly address an emerging controversy relating to the DNA architecture in the maturation complex that mediates complex stability. We next test the hypothesis that the lambda motor also functions as a tetrameric complex and that ATP hydrolysis by the motor is strongly cooperative; these features represent a significant departure from currently accepted paradigms. Finally, we characterize a putative nucleotide switch mechanism that controls the transition from the stable maturation complex to the dynamic motor complex bound to the capsid and we rigorously define the energy budget of the translocating motor. The proposed studies will provide structural and mechanistic detail on two sequential packaging complexes and their transition through the genome-packaging pathway. These features are shared by all of the dsDNA viruses that package genomes from concatemeric precursors (phage, herpes) and the results will be of broad and general significance.