Bacteriophage PM2 has a protein capsid surrounding a spherical proteinaceous lipid core

The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae, infecting two gram-negative Pseudoalteromonas species. The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins. The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer. The C-terminal part of P1 is distal and is responsible for receptor binding activity. The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome. This internal particle is designated the lipid core. The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family TECTIVIRIDAE:     

Mechanism of Membranous Tunnelling Nanotube Formation in Viral Genome Delivery

In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.     

Membrane-assisted viral DNA ejection

Genome packaging and delivery are fundamental steps in the replication cycle of all viruses. Icosahedral viruses with linear double-stranded DNA (dsDNA) usually package their genome into a preformed, rigid procapsid using the power generated by a virus-encoded packaging ATPase. The pressure and stored energy due to this confinement of DNA at a high density is assumed to drive the initial stages of genome ejection. Membrane-containing icosahedral viruses, such as bacteriophage PRD1, present an additional architectural complexity by enclosing their genome within an internal membrane vesicle. Upon adsorption to a host cell, the PRD1 membrane remodels into a proteo-lipidic tube that provides a conduit for passage of the ejected linear dsDNA through the cell envelope. Based on volume analyses of PRD1 membrane vesicles captured by cryo-electron tomography and modeling of the elastic properties of the vesicle, we propose that the internal membrane makes a crucial and active contribution during infection by maintaining the driving force for DNA ejection and countering the internal turgor pressure of the host. These novel functions extend the role of the PRD1 viral membrane beyond tube formation or the mere physical confinement of the genome. The presence and assistance of an internal membrane might constitute a biological advantage that extends also to other viruses that package their linear dsDNA to high density within an internal vesicle.     

Genetics for Pseudoalteromonas provides tools to manipulate marine bacterial virus PM2

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (Corticoviridae) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative Pseudoalteromonas species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.     

The PM2 virion has a novel organization with an internal membrane and pentameric receptor binding spikes

Biological membranes are notoriously resistant to structural analysis. Excellent candidates to tackle this problem in situ are membrane-containing viruses where the membrane is constrained by an icosahedral capsid. Cryo-EM and image reconstruction of bacteriophage PM2 revealed a membrane bilayer following the internal surface of the capsid. The viral genome closely interacts with the inner leaflet. The capsid, at a resolution of 8.4 A, reveals 200 trimeric capsomers with a pseudo T = 21 dextro organization. Pentameric receptor-binding spikes protrude from the surface. It is evident from the structure that the PM2 membrane has at least two important roles in the life cycle. First, it acts as a scaffold to nucleate capsid assembly. Second, after host recognition, it fuses with the host outer membrane to promote genome entry. The structure also sheds light on how the viral supercoiled circular double-stranded DNA genome might be packaged and released.     

An ssDNA virus infecting archaea: a new lineage of viruses with a membrane envelope

Archaeal organisms are generally known as diverse extremophiles, but they play a crucial role also in moderate environments. So far, only about 50 archaeal viruses have been described in some detail. Despite this, unusual viral morphotypes within this group have been reported. Interestingly, all isolated archaeal viruses have a double-stranded DNA (dsDNA) genome. To further characterize the diversity of archaeal viruses, we screened highly saline water samples for archaea and their viruses. Here, we describe a new haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1) that was isolated from a solar saltern and infects an indigenous host belonging to the genus Halorubrum . Infection does not cause cell lysis, but slightly retards growth of the host and results in high replication of the virus. The sequenced genome (7048 nucleotides) of HRPV-1 is single-stranded DNA (ssDNA), which makes HRPV-1 the first characterized archaeal virus that does not have a dsDNA genome. In spite of this, similarities to another archaeal virus were observed. Two major structural proteins were recognized in protein analyses, and by lipid analyses it was shown that the virion contains a membrane. Electron microscopy studies indicate that the enveloped virion is pleomorphic (approximately 44 × 55 nm). HRPV-1 virion may represent commonly used virion architecture, and it seems that structure-based virus lineages may be extended to non-icosahedral viruses.     

Structure and assembly of bacteriophage PRD1, and Escherichia coli virus with a membrane

This article describes the structure and assembly of bacteriophage PRD1, a lipid-containing virus able to infect Escherichia coli. This phage, with an approximate diameter of 65 nm, is composed of an outer protein shell surrounding a lipid-protein membrane which, in turn, encloses the nucleic acid. The phage genome consists of a single linear dsDNA molecule of about 15 kb that has a protein covalently linked to each of its 5' ends. This protein is used as a primer in DNA replication. During assembly membrane proteins are inserted into the host cytoplasmic membrane while major capsid protein multimers are found in the cytoplasm. Capsid multimers, assisted by two nonstructural assembly factors, are capable of translocating the virus-specific membrane resulting in the formation of cytoplasmic empty particles. Subsequent DNA packaging leads to the formation of infections virus.     

Structure and assembly of bacteriophage PRD1, an Escherichia coli virus with a membrane

This article describes the structure and assembly of bacteriophage PRD1, a lipid-containing virus able to infect Escherichia coli. This phage, with an approximate diameter of 65 nm, is composed of an outer protein shell surrounding a lipid-protein membrane which, in turn, encloses the nucleic acid. The phage genome consists of a single linear dsDNA molecule of about 15 kb that has a protein covalently linked to each of its 5′ ends. This protein is used as a primer in DNA replication. During assembly membrane proteins are inserted into the host cytoplasmic membrane while major capsid protein multimers are found in the cytoplasm. Capsid multimers, assisted by two nonstructural assembly factors, are capable of translocating the virus-specific membrane resulting in the formation of cytoplasmic empty particles. Subsequent DNA packaging leads to the formation of infectious virus.     

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 Bridge Crosses the Active-Site Canyon of the Epstein-Barr Virus Nuclease with DNase and RNase Activities

Epstein-Barr virus, a double-stranded DNA (dsDNA) virus, is a major human pathogen from the herpesvirus family. The nuclease is one of the lytic cycle proteins required for successful viral replication. In addition to the previously described endonuclease and exonuclease activities on single-stranded DNA and dsDNA substrates, we observed an RNase activity for Epstein-Barr virus nuclease in the presence of Mn²⁺, giving a possible explanation for its role in host mRNA degradation. Its crystal structure shows a catalytic core of the D-(D/E)XK nuclease superfamily closely related to the exonuclease from bacteriophage lambda with a bridge across the active-site canyon. This bridge may reduce endonuclease activity, ensure processivity or play a role in strand separation of dsDNA substrates. As the DNA strand that is subject to cleavage is likely to make a sharp turn in front of the bridge, endonuclease activity on single-stranded DNA stretches appears to be possible, explaining the cleavage of circular substrates.     

A Structural Model of the Genome Packaging Process in a Membrane-Containing Double Stranded DNA Virus

Two crucial steps in the virus life cycle are genome encapsidation to form an infective virion and genome exit to infect the next host cell. In most icosahedral double-stranded (ds) DNA viruses, the viral genome enters and exits the capsid through a unique vertex. Internal membrane-containing viruses possess additional complexity as the genome must be translocated through the viral membrane bilayer. Here, we report the structure of the genome packaging complex with a membrane conduit essential for viral genome encapsidation in the tailless icosahedral membrane-containing bacteriophage PRD1. We utilize single particle electron cryo-microscopy (cryo-EM) and symmetry-free image reconstruction to determine structures of PRD1 virion, procapsid, and packaging deficient mutant particles. At the unique vertex of PRD1, the packaging complex replaces the regular 5-fold structure and crosses the lipid bilayer. These structures reveal that the packaging ATPase P9 and the packaging efficiency factor P6 form a dodecameric portal complex external to the membrane moiety, surrounded by ten major capsid protein P3 trimers. The viral transmembrane density at the special vertex is assigned to be a hexamer of heterodimer of proteins P20 and P22. The hexamer functions as a membrane conduit for the DNA and as a nucleating site for the unique vertex assembly. Our structures show a conformational alteration in the lipid membrane after the P9 and P6 are recruited to the virion. The P8-genome complex is then packaged into the procapsid through the unique vertex while the genome terminal protein P8 functions as a valve that closes the channel once the genome is inside. Comparing mature virion, procapsid, and mutant particle structures led us to propose an assembly pathway for the genome packaging apparatus in the PRD1 virion.     

Multiple consecutive initiation of replication producing novel brush-like intermediates at the termini of linear viral dsDNA genomes with hairpin ends

Linear dsDNA replicons with hairpin ends are found in the three domains of life, mainly associated with plasmids and viruses including the poxviruses, some phages and archaeal rudiviruses. However, their replication mechanism is not clearly understood. In this study, we find that the rudivirus SIRV2 undergoes multiple consecutive replication reinitiation events at the genomic termini. Using a strand-displacement replication strategy, the multiple reinitiation events from one parental template yield highly branched intermediates corresponding to about 30 genome units which generate exceptional ‘brush-like’ structures. Moreover, our data support the occurrence of an additional strand-coupled bidirectional replication from a circular dimeric intermediate. The multiple reinitiation process ensures rapid copying of the parental viral genome and will enable protein factors involved in viral genome replication to be specifically localised intracellularly, thereby helping the virus to avoid host defence mechanisms.     

The structure of the ATPase that powers DNA packaging into bacteriophage T4 procapsids

Packaging the viral genome into empty procapsids, an essential event in the life cycle of tailed bacteriophages and some eukaryotic viruses, is a process that shares features with chromosome assembly. Most viral procapsids possess a special vertex containing a dodecameric portal protein that is used for entry and exit of the viral genome. The portal and an ATPase are parts of the genome-packaging machine. The ATPase is required to provide energy for translocation and compaction of the negative charges on the genomic DNA. Here we report the atomic structure of the ATPase component in a phage DNA-packaging machine. The bacteriophage T4 ATPase has the greatest similarity to monomeric helicases, suggesting that the genome is translocated by an inchworm mechanism. The similarity of the packaging machines in the double-stranded DNA (dsDNA) bacteriophage T4 and dsRNA bacteriophage varphi12 is consistent with the evolution of many virions from a common ancestor.     

Constituents of SH1, a novel lipid-containing virus infecting the halophilic euryarchaeon Haloarcula hispanica

Recent studies have indicated that a number of bacterial and eukaryotic viruses that share a common architectural principle are related, leading to the proposal of an early common ancestor. A prediction of this model would be the discovery of similar viruses that infect archaeal hosts. Our main interest lies in icosahedral double-stranded DNA (dsDNA) viruses with an internal membrane, and we now extend our studies to include viruses infecting archaeal hosts. While the number of sequenced archaeal viruses is increasing, very little sequence similarity has been detected between bacterial and eukaryotic viruses. In this investigation we rigorously show that SH1, an icosahedral dsDNA virus infecting Haloarcula hispanica, possesses lipid structural components that are selectively acquired from the host pool. We also determined the sequence of the 31-kb SH1 genome and positively identified genes for 11 structural proteins, with putative identification of three additional proteins. The SH1 genome is unique and, except for a few open reading frames, shows no detectable similarity to other published sequences, but the overall structure of the SH1 virion and its linear genome with inverted terminal repeats is reminiscent of lipid-containing dsDNA bacteriophages like PRD1.     

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. Analysis of the reaction suggests that (i) virus assembly in vitro is optimal under conditions that faithfully mimic the intracellular environment within an Escherichia coli cell, (ii) concatemeric DNA is required for the successful completion of virus assembly, (iii) several of the protein components oligomerize concomitant with their step-wise addition to the nascent virus particle and (iv) tail addition is the rate-limiting step in virus assembly. Importantly, the assembled virus may enter either of the developmental pathways (lytic or lysogenic) expected of a lambda virion. Thus, we demonstrate for the first time that a wild-type, complex DNA virus may be assembled from purified components under defined biochemical conditions. This system provides a powerful tool to characterize, at the molecular level, the step-by-step processes required to assemble an infectious virus particle. Given the remarkable similarities between dsDNA bacteriophage and eukaryotic dsDNA viruses, characterization of the lambda system has broad biological implications in our understanding of virus development at a global level.     

Integration and expression of several molecular forms of Rous sarcoma virus DNA used for transfection of mouse cells.

To assess the factors required for integration and expression of retroviral DNA, we have examined viral DNA, RNA, and protein in NIH/3T3 mouse cells transformed by transfection with various forms of cloned Rous sarcoma virus (RSV) DNA. Linear RSV DNA molecules, derived from circular DNA containing two long terminal repeats (LTRs) and permuted by cleavage at the SacI restriction endonuclease site in the leader sequence, were integrated near the ends of the linear molecule, with the LTRs on the 3' side of the src gene. Integration of a subgenomic RSV DNA fragment containing the viral src gene without intact LTRs also occurred near the ends of the linear molecule. Head-to-tail tandem arrays of RSV DNA species were observed in some transformed cell lines that received fully digested DNA and in all cell lines that received DNA ligated to produce oligomers before transfection. Closed circular RSV DNA, with one or two LTRs, integrated without apparent specificity within several regions of the viral genome. After transfection with SacI-permuted RSV DNA still linked to arms of the lambda bacteriophage vector DNA, bacteriophage sequences were joined to host DNA. Transformed cell lines produced by transfection with the various forms of RSV DNA produced similar levels of viral src protein, although the efficiency of successful transformation varied by at least two orders of magnitude. Analyses of viral polyadenylated RNA, together with the patterns of viral DNA in transformed cells, indicated that viral DNA can be integrated and expressed without regard to LTR sequences, with adjacent host DNA presumably supplying signals required for the promotion and processing of functional src mRNA.     

Target Membrane Cholesterol Modulates Single Influenza Virus Membrane Fusion Efficiency but Not Rate

Host lipid composition influences many stages of the influenza A virus (IAV) entry process, including initial binding of IAV to sialylated glycans, fusion between the viral envelope and the host membrane, and the formation of a fusion pore through which the viral genome is transferred into a target cell. In particular, target membrane cholesterol has been shown to preferentially associate with virus receptors and alter physical properties of the membrane like fluidity and curvature. These properties affect both IAV binding and fusion, which makes it difficult to isolate the role of cholesterol in IAV fusion from receptor binding effects. Here, we develop a fusion assay that uses synthetic DNA-lipid conjugates as surrogate viral receptors to tether virions to target vesicles. To avoid the possibly perturbative effect of adding a self-quenched concentration of dye-labeled lipids to the viral membrane, we tether virions to lipid-labeled target vesicles and use fluorescence microscopy to detect individual, pH-triggered IAV membrane fusion events. Through this approach, we find that cholesterol in the target membrane enhances the efficiency of single-particle IAV lipid mixing, whereas the rate of lipid mixing is independent of cholesterol composition. We also find that the single-particle kinetics of influenza lipid mixing to target membranes with different cholesterol compositions is independent of receptor binding, suggesting that cholesterol-mediated spatial clustering of viral receptors within the target membrane does not significantly affect IAV hemifusion. These results are consistent with the hypothesis that target membrane cholesterol increases lipid mixing efficiency by altering host membrane curvature.     

Membrane-containing viruses with icosahedrally symmetric capsids

Viruses with an icosahedrally symmetric protein capsid and a membrane infect hosts from all three domains of life. Similar architectural principles are shared by different viral families, as exemplified by double-stranded DNA viruses such as PRD1 and STIV. During virus assembly, the membrane lipids are selectively acquired from the host cell. The X-ray structure of bacteriophage PRD1 revealed that the lipids are asymmetrically distributed between the two leaflets and facet length is controlled by a tape-measure protein. In most membrane-containing viruses, viral and host membranes fuse during viral entry. In the best-understood systems of the alphaviruses, flaviviruses and herpes viruses, fusion is mediated by viral glycoproteins. Recent structural advances reveal how very different protein architectures can be used to form trimeric extensions that extend into the target cell membrane and then fold back to mediate fusion of the target and viral membranes.     

Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion

In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.     

Structural determinants of the interaction between influenza A virus matrix protein M1 and lipid membranes

Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten major proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1–protein interactions and multimerization have not been clarified, yet.In this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95–105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.