Translation of the long-term fundamental studies on viral DNA packaging motors into nanotechnology and nanomedicine

Many years of fundamental studies on viral genome packaging motors have led to fruitful applications. The double-stranded DNA(dsDNA) viruses package their genomes into preformed protein shells via nanomotors including several elegant and meticulous coaxial modules. The motor is geared by the hexameric RNA ring. An open washer displayed as hexametric string of phi29 motor ATPase has been reported. The open washer linked into a filament as a queue with left-handed chirality along the dsDNA chain. It was found that a free 5′-and 3′-dsDNA end is not required for one gp16 dimer and four monomers to assemble into the hexametric washer on dsDNA. The above studies have inspired several applications in nanotechnology and nanomedicine. These applications include:(i) studies on the precision motor channels have led to their application in the single pore sensing;(ii) investigations into the hand-in-hand integration of the hexametric pRNA ring have resulted in the emergence of the new field of RNA nanotechnology; and(iii) the studies on the motor stoichiometry of homologous multi-subunits that subsequently have inspired the discovery of new methods in highly potent drug development. This review focuses on the structure and function of the viral DNA packaging motors and describes how fundamental studies inspired various applications. Given these advantages, more nanotechnological and biomedical applications using bacteriophage motor components are expected.     

Biological and biochemical properties of the small viral RNA(pRNA) essential for the packaging of the dsDNA of phage ø29

The genome of the lineal double-stranded DNA viruses of both prokaryotes and eukaryotes is packaged into a preformed procapsid during maturation. Common features exist in this step of the viral life cycle. Bacteriophage ø29 is an ideal model in this study because its DNA can be efficiently packaged in vitro with all components overproduced and purified. An exciting aspect is the discovery that a small viral RNA (pRNA) encoded by ø29 has a novel and essential role in viral DNA packaging. This pRNA is not a structural component of the mature virion, nor is it required for the assembly of the procapsid. The discovery of pRNA as a non-protein participant in viral DNA packaging extends previously demonstrated RNA functions.     

Evaluation of end-capped DNA modules for pRNA capture and functionalization

The ability of packaging RNA (pRNA) from the phi29 DNA packaging motor to form nanoassemblies and nanostructures has been exploited for the development of the nascent field of RNA nanotechnology and subsequent applications in nanomedicine. For applications in nanomedicine, it is necessary to modify the pRNA structure for the conjugation of active molecules. We have investigated end-capped double-stranded DNA segments as reversible capture reagents for pRNA. These capture agents can be designed to allow the conjugation of any desired molecule for pRNA functionalization. The results of model studies presented in this report show that 5- to 7-nucleotide overhangs on a target RNA can provide efficient handles for the high-affinity association to capped double-stranded DNA.     

RNA packaging device of double-stranded RNA bacteriophages,possibly as simple as hexamer of P4 protein

Genomes of complex viruses have been demonstrated, in many cases, to be packaged into preformed empty capsids (procapsids). This reaction is performed by molecular motors translocating nucleic acid against the concentration gradient at the expense of NTP hydrolysis. At present, the molecular mechanisms of packaging remain elusive due to the complex nature of packaging motors. In the case of the double-stranded RNA bacteriophage phi 6 from the Cystoviridae family, packaging of single-stranded genomic precursors requires a hexameric NTPase, P4. In the present study, the purified P4 proteins from two other cystoviruses, phi 8 and phi 13, were characterized and compared with phi 6 P4. All three proteins are hexameric, single-stranded RNA-stimulated NTPases with alpha/beta folds. Using a direct motor assay, we found that phi 8 and phi 13 P4 hexamers translocate 5' to 3' along ssRNA, whereas the analogous activity of phi 6 P4 requires association with the procapsid. This difference is explained by the intrinsically high affinity of phi 8 and phi 13 P4s for nucleic acids. The unidirectional translocation results in RNA helicase activity. Thus, P4 proteins of Cystoviridae exhibit extensive similarity to hexameric helicases and are simple models for studying viral packaging motor mechanisms.     

Viral enzymes

Viral genomes show unequalled diversity, ranging from single-stranded DNA to double-stranded RNA. Moreover, viruses can quickly adapt to the host's immune response and drug treatment. Although they tend to make optimal use of the host cell's reservoir of proteins, viruses need to carry some enzymatic functions with them, as they may not be available or accessible in the infected cell. Recently, progress has been made in our structural understanding of viral enzymes involved in all stages of the viral life cycle, which includes entry, hijack, replication and exit stages.     

Viral evolution and insects as a possible virologic turning table

Summary Three lines of observation demonstrate the role of arthropods in transmission and evolution of viruses. a) Recent outbreaks of viruses from their niches took place and insects have played a major role in propagating the viruses. b) Examination of the list of viral families and their hosts shows that many infect invertebrates (I) and vertebrates (V) or (I) and plants (P) or all kingdoms (VIPs). This notion holds true irrespective of the genome type. At first glance the argument seems to be weak in the case of enveloped and non-enveloped RNA viruses with single-stranded (ss) segmented or non-segmented genomes of positive (+) or negative polarity. Here, there are several families infecting V or P only; no systematic relation to arthropods is found. c) In the non-enveloped plant viruses with ss RNA genomes there is a strong tendency for segmentation and individual packaging of the genome pieces. This is in contrast to ss+ RNA animal viruses and can only be explained by massive transmission by seed or insects or both, because individual packaging necessitates a multihit infection. Comparisons demonstrate relationships in the nonstructural proteins of double-stranded and ss+ RNA viruses irrespective of host range, segmentation, and envelope. Similar conclusions apply for the negative-stranded RNA viruses. Thus, viral supergroups can be created that infect V or P and exploit arthropods for infection or transmission or both. Examples of such relationships and explanations for viral evolution are reviewed and the arthropod orders important for cell culture are given.     

Characterization of the archaeal thermophile Sulfolobus turreted icosahedral virus validates an evolutionary link among double-stranded DNA viruses from all domains of life

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.     

A promiscuous DNA packaging machine from bacteriophage T4

Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.     

In vitro DNA packaging of PRD1: a common mechanism for internal-membrane viruses

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.     

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 ∼ 20 atm of internal capsid pressure. Herein we describe biochemical studies on genome packaging using bacteriophage λ as a model system. Kinetic analysis suggests that the packaging motor possesses at least four ATPase catalytic sites that act cooperatively to effect DNA translocation, and that the motor is highly processive. While not required for DNA translocation into the capsid, the phage λ capsid decoration protein gpD is essential for the packaging of the penultimate 8-10 kb (15-20%) of the viral genome; virtually no DNA is packaged in the absence of gpD when large DNA substrates are used, most likely due to a loss of capsid structural integrity. Finally, we show that ATP hydrolysis is required to retain the genome in a packaged state subsequent to condensation within the capsid. Presumably, the packaging motor continues to “idle” at the genome end and to maintain a positive pressure towards the packaged state. Surprisingly, ADP, guanosine triphosphate, and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) similarly stabilize the packaged viral genome despite the fact that they fail to support genome packaging. In contrast, the poorly hydrolyzed ATP analog ATP-γS only partially stabilizes the nucleocapsid, and a DNA is released in “quantized” steps. We interpret the ensemble of data to indicate that (i) the viral procapsid possesses a degree of plasticity that is required to accommodate the packaging of large DNA substrates; (ii) the gpD decoration protein is required to stabilize the fully expanded capsid; and (iii) nucleotides regulate high-affinity DNA binding interactions that are required to maintain DNA in the packaged state.     

Distinct DNA exit and packaging portals in the virus Acanthamoeba polyphaga mimivirus

Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the “stargate”, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses.     

Distinct DNA Exit and Packaging Portals in the Virus Acanthamoeba polyphaga mimivirus

Icosahedral double-stranded DNA viruses use a single portal for genome delivery and packaging. The extensive structural similarity revealed by such portals in diverse viruses, as well as their invariable positioning at a unique icosahedral vertex, led to the consensus that a particular, highly conserved vertex-portal architecture is essential for viral DNA translocations. Here we present an exception to this paradigm by demonstrating that genome delivery and packaging in the virus Acanthamoeba polyphaga mimivirus occur through two distinct portals. By using high-resolution techniques, including electron tomography and cryo-scanning electron microscopy, we show that Mimivirus genome delivery entails a large-scale conformational change of the capsid, whereby five icosahedral faces open up. This opening, which occurs at a unique vertex of the capsid that we coined the “stargate”, allows for the formation of a massive membrane conduit through which the viral DNA is released. A transient aperture centered at an icosahedral face distal to the DNA delivery site acts as a non-vertex DNA packaging portal. In conjunction with comparative genomic studies, our observations imply a viral packaging pathway akin to bacterial DNA segregation, which might be shared by diverse internal membrane–containing viruses.     

Biosynthesis of virus-specific proteins in cells infected with infectious bursal disease virus and their significance as structural elements for infectious virus and incomplete particles.

It has previously been shown that infectious bursal disease virus is a naked icosahedral particle with a diameter of about 60 nm and a genome consisting of two segments of double-stranded RNA (Müller et al., J. Virol. 31:584-589, 1979). One of the two major structural polypeptides (molecular weight, 40,000) of this virus could not be found in lysates of infected cells; it is derived from a precursor polypeptide demonstrable inside the cells in relatively large quantities and seems to be processed during virus assembly or later. The precursor molecule is regularly present in the infectious virus particle (buoyant density, 1.33 g/ml) in minor proportions, but it represents an outstanding structural element of incomplete noninfectious particles ("top components"; buoyant density, 1.29 g/ml) which contain viral RNA. This type of incomplete particles is mainly produced by chicken embryo fibroblasts in contrast to lymphoid cells from the bursa of Fabricius. Precursor-product relationships also seem to exist in the biosynthesis of the other viral polypeptides. In contrast to some other viruses with a segmented double-stranded RNA genome, none of the structural proteins of infectious bursal disease virus is appreciably glycosylated.     

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. coli integration host factor protein (IHF). Here, we show that the protomer further assembles into a homogeneous tetramer of protomers of composition (gpA1/gpNu1(2))4. Electron microscopy shows that the tetramer forms a ring structure large enough to encircle duplex DNA. In contrast to the heterotrimer, the ring tetramer can mature and package viral DNA in the absence of IHF. We propose that IHF induced bending of viral DNA facilitates the assembly of four terminase protomers into a ring tetramer that represents the catalytically competent DNA maturation and packaging complex in vivo. This work provides, for the first time, insight into the functional assembly state of a viral DNA packaging motor.     

Mechanisms of human immunodeficiency virus type 2 RNA packaging: efficient trans packaging and selection of RNA copackaging partners

Human immunodeficiency virus type 2 (HIV-2) has been reported to have a distinct RNA packaging mechanism, referred to as cis packaging, in which Gag proteins package the RNA from which they were translated. We examined the progeny generated from dually infected cell lines that contain two HIV-2 proviruses, one with a wild-type gag/gag-pol and the other with a mutant gag that cannot express functional Gag/Gag-Pol. Viral titers and RNA analyses revealed that mutant viral RNAs can be packaged at efficiencies comparable to that of viral RNA from which wild-type Gag/Gag-Pol is translated. These results do not support the cis-packaging hypothesis but instead indicate that trans packaging is the major mechanism of HIV-2 RNA packaging. To further characterize the mechanisms of HIV-2 RNA packaging, we visualized HIV-2 RNA in individual particles by using fluorescent protein-tagged RNA-binding proteins that specifically recognize stem-loop motifs in the viral genomes, an assay termed single virion analysis. These studies revealed that >90% of the HIV-2 particles contained viral RNAs and that RNAs derived from different viruses were copackaged frequently. Furthermore, the frequencies of heterozygous particles in the viral population could be altered by changing a 6-nucleotide palindromic sequence at the 5'-untranslated region of the HIV-2 genome. This finding indicates that selection of copackaging RNA partners occurs prior to encapsidation and that HIV-2 Gag proteins primarily package one dimeric RNA rather than two monomeric RNAs. Additionally, single virion analyses demonstrated a similar RNA distribution in viral particles regardless of whether both viruses had a functional gag or one of the viruses had a nonfunctional gag, providing further support for the trans-packaging hypothesis. Together, these results revealed mechanisms of HIV-2 RNA packaging that are, contrary to previous studies, in many respects surprisingly similar to those of HIV-1.     

Viral connectors for DNA encapsulation

Viral connectors are key components of the life cycle of bacteriophages and other viral systems. They participate in procapsid assembly, and they are instrumental in DNA packaging and release. Connector proteins build hollow cylindrical dodecamers that show an overall morphological similarity among different viral systems including a remarkable conserved domain in the central part of the protein. These domains build the wall of the channel forming a 24 α-helices stretch together with an α-β extension. A similar α-helical arrangement is found in other unspecific DNA translocating complexes, suggesting the existence of a common structural signature for channel formation. Preliminary experiments suggest that connectors might be ideal candidates as nanopores for synthetic applications in nanotechnology.     

Mechanisms of nucleic acid translocases: lessons from structural biology and single-molecule biophysics

Enzymes that translocate nucleic acids using ATP hydrolysis include DNA and RNA helicases, viral genome packaging motors and chromatin remodeling ATPases. Recent structural analysis, in conjunction with single-molecule studies, has revealed a wealth of new insights into how these enzymes use ATP-driven conformational changes to move on nucleic acids.     

Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a genome-packaging motor

Terminase enzymes are common to both prokaryotic and eukaryotic double-stranded DNA viruses and are responsible for packaging viral DNA into the confines of an empty procapsid shell. In all known cases, the holoenzymes are heteroligomers composed of a large subunit that possesses the catalytic activities required for genome packaging and a small subunit that is responsible for specific recognition of viral DNA. In bacteriophage lambda, the DNA recognition protein is gpNu1. The gpNu1 subunit interacts with multiple recognition elements within cos, the packaging initiation site in viral DNA, to site-specifically assemble the packaging machinery. Motor assembly is modulated by the Escherichia coli integration host factor protein (IHF), which binds to a consensus sequence also located within cos. On the basis of a variety of biochemical data and the recently solved NMR structure of the DNA binding domain of gpNu1, we proposed a novel DNA binding mode that predicts significant bending of duplex DNA by gpNu1 (de Beer et al. (2002) Mol. Cell 9, 981-991). We further proposed that gpNu1 and IHF cooperatively bind and bend viral DNA to regulate the assembly of the packaging motor. Here, we characterize cooperative gpNu1 and IHF binding to the cos site in lambda DNA using a quantitative electrophoretic mobility shift (EMS) assay. These studies provide direct experimental support for the long presumed cooperative assembly of gpNu1 and IHF at the cos sequence of lambda DNA. Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. Thus, specific recognition of viral DNA by the packaging apparatus is mediated by both DNA sequence information and by structural alteration of the duplex. The relevance of these results with respect to the assembly of a viral DNA-packaging motor is discussed.     

Measurements of single DNA molecule packaging dynamics in bacteriophage lambda reveal high forces, high motor processivity, and capsid transformations

Molecular motors drive genome packaging into preformed procapsids in many double-stranded (ds)DNA viruses. Here, we present optical tweezers measurements of single DNA molecule packaging in bacteriophage lambda. DNA-gpA-gpNu1 complexes were assembled with recombinant gpA and gpNu1 proteins and tethered to microspheres, and procapsids were attached to separate microspheres. DNA binding and initiation of packaging were observed within a few seconds of bringing these microspheres into proximity in the presence of ATP. The motor was observed to generate greater than 50 picoNewtons (pN) of force, in the same range as observed with bacteriophage phi29, suggesting that high force generation is a common property of viral packaging motors. However, at low capsid filling the packaging rate averaged approximately 600 bp/s, which is 3.5-fold higher than phi29, and the motor processivity was also threefold higher, with less than one slip per genome length translocated. The packaging rate slowed significantly with increasing capsid filling, indicating a buildup of internal force reaching 14 pN at 86% packaging, in good agreement with the force driving DNA ejection measured in osmotic pressure experiments and calculated theoretically. Taken together, these experiments show that the internal force that builds during packaging is largely available to drive subsequent DNA ejection. In addition, we observed an 80 bp/s dip in the average packaging rate at 30% packaging, suggesting that procapsid expansion occurs at this point following the buildup of an average of 4 pN of internal force. In experiments with a DNA construct longer than the wild-type genome, a sudden acceleration in packaging rate was observed above 90% packaging, and much greater than 100% of the genome length was translocated, suggesting that internal force can rupture the immature procapsid, which lacks an accessory protein (gpD).