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SummaryThe HIV-1 entry inhibitor temsavir prevents CD4 from interacting with the envelope glycoprotein (Env) and blocks its conformational changes. To do this temsavir relies on the presence of a residue with small side chain at position 375 in Env and is unable to neutralize viral strains like CRF01_AE carrying His375. Here we investigate the mechanism of temsavir-resistance and show that residue 375 is not the sole determinant of resistance. At least six additional residues within the gp120 inner domain layers, including five distant from the drug-binding pocket, contribute to resistance. A detailed structure-function analysis using engineered viruses and soluble trimer variants reveal that the molecular basis of resistance is mediated by crosstalk between His375 and the inner domain layers. Furthermore, our data confirm that temsavir can adjust its binding mode to accommodate changes in Env conformation, a property that likely contributes to its broad-antiviral activity.

Many icosahedral viruses including tailed bacteriophages and herpes viruses have a unique portal vertex where a dodecameric protein ring is associated with a fivefold capsid shell. While the peripheral regions of the portal ring are involved in capsid assembly, its central channel is used to transport DNA into and out of capsid during genome packaging and infection. Though the atomic structure of this highly conserved, turbine-shaped, portal is known for nearly two decades, its molecular mechanism remains a mystery. Recent high-resolution in situ structures reveal various conformational states of the portal and the asymmetric interactions between the 12-fold portal and the fivefold capsid. These lead to a valve-like mechanism for this symmetry-mismatched portal vertex that regulates DNA flow through the channel, a critical function for high fidelity assembly of an infectious virion.

Bacteriophage T4 has enormous potential for biomedical applications due to its large size, capsid architecture, and high payload capability for protein and DNA delivery. However, it is not very easy to genetically engineer its genome heavily modified by cytosine hydroxymethylation and glucosylation. The glucosyl hydroxymethyl cytosine (ghmC) genome of phage is completely resistant to most restriction endonucleases and exhibits various degrees of resistance to CRISPR-Cas systems. Here, we found that the type V CRISPR-Cas12a system, which shows efficient cleavage of ghmC-modified genome when compared to the type II CRISPR-Cas9 system, can be synergistically employed to generate recombinant T4 phages. Focused on surface display, we analyzed the ability of phage T4 outer capsid proteins Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein) to tether, in vivo, foreign peptides and proteins to T4 capsid. Our data show that while these could be successfully expressed and displayed during the phage infection, shorter peptides are present at a much higher copy number than full-length proteins. However, the copy number of the latter could be elevated by driving the expression of the transgene using the strong T7 RNA polymerase expression system. This CRISPR-inspired approach has the potential to expand the application of phages to various basic and translational research projects.

Many icosahedral viruses assemble proteinaceous precursors called proheads or procapsids. Proheads are metastable structures that undergo a profound structural transition known as expansion that transforms an immature unexpanded head into a mature genome-packaging head. Bacteriophage T4 is a model virus, well studied genetically and biochemically, but its structure determination has been challenging because of its large size and unusually prolate-shaped, ∼1,200-Å-long and ∼860-Å-wide capsid. Here, we report the cryogenic electron microscopy (cryo-EM) structures of T4 capsid in both of its major conformational states: unexpanded at a resolution of 5.1 Å and expanded at a resolution of 3.4 Å. These are among the largest structures deposited in Protein Data Bank to date and provide insights into virus assembly, head length determination, and shell expansion. First, the structures illustrate major domain movements and ∼70% additional gain in inner capsid volume, an essential transformation to contain the entire viral genome. Second, intricate intracapsomer interactions involving a unique insertion domain dramatically change, allowing the capsid subunits to rotate and twist while the capsomers remain fastened at quasi-threefold axes. Third, high-affinity binding sites emerge for a capsid decoration protein that clamps adjacent capsomers, imparting extraordinary structural stability. Fourth, subtle conformational changes at capsomers’ periphery modulate intercapsomer angles between capsomer planes that control capsid length. Finally, conformational changes were observed at the symmetry-mismatched portal vertex, which might be involved in triggering head expansion. These analyses illustrate how small changes in local capsid subunit interactions lead to profound shifts in viral capsid morphology, stability, and volume.

The ability to deliver therapeutic genes and biomolecules into a human cell and restore a defective function has been the holy grail of medicine. Adeno-associated viruses and lentiviruses have been extensively used as delivery vehicles, but their capacity is limited to one (or two) gene(s). Bacteriophages are emerging as novel vehicles for gene therapy. The large 120 × 86-nm T4 capsid allows engineering of both its surface and its interior to incorporate combinations of DNAs, RNAs, proteins, and their complexes. In vitro assembly using purified components allows customization for various applications and for individualized therapies. Its large capacity, cell-targeting capability, safety, and inexpensive manufacturing could open unprecedented new possibilities for gene, cancer, and stem cell therapies. However, efficient entry into primary human cells and intracellular trafficking are significant barriers that must be overcome by gene engineering and evolution in order to translate phage-delivery technology from bench to bedside.

The interplay between defense and counter-defense systems of bacteria and phages is a major driver of evolution of both organisms, leading to their greatest genetic diversity. Bacterial restriction-modification (R-M) and CRISPR-Cas are two well-known defense systems that target phage DNAs through their nuclease activities, whereas phages have developed counter-defense systems through covalent modifications of their genomes. Recent studies have revealed many novel nuclease-containing antiphage systems, which leads to the question of what’s the role of phage genome modifications in countering these systems. Here, we scanned Escherichia coli genome sequences available in the NCBI databases and found abundant nuclease-containing defense systems, indicating that phage genomic DNA could be a major target for E. coli to restrict infection. From a collection of 816 E. coli strains, we cloned and validated 14 systems. Particularly, Gabija and type III Druantia systems have broad antiphage activities. Using wild-type phage T4 and its mutants, T4 (hmC) and T4 (C), which contain glucosyl-5-hydroxymethylcytosines, 5-hydroxymethylcytosines, and unmodified cytosines in the genomic DNA respectively, we revealed the complex roles of genomic modification of phage T4 in countering the nuclease-containing defense systems other than simply blocking the degradation of genomic DNA by nuclease.

Mucosal vaccines that can induce local mucosal immune responses and combat the pathogens at entry sites are considered to be the most effective way to prevent infection. A universal platform that can be customized for development of mucosal vaccines against any given pathogen is therefore highly desired. Here, we demonstrate an efficient approach to develop nasal mucosal vaccines through genetic engineering of T4 phage to generate antigen-decorated nanoparticles. The antigen coding sequence was inserted into T4 genome in-frame at the C terminus of Soc (small outer capsid protein) using the CRISPR-Cas phage editing technology. During the propagation of recombinant T4 phages in E. coli, the Soc-antigen fusion proteins self-assemble on T4 capsids to form antigen-decorated nanoparticles that have intrinsic adjuvant activity and mucosal adhesive property. As a proof of concept, we showed that intranasal immunization with Flu viral M2e-decorated T4 nanoparticles efficiently induced local mucosal as well as systemic immune responses and provided complete protections against divergent influenza viruses in a mouse model. Potentially, our platform can be customized for any respiratory pathogen to rapidly generate mucosal vaccines against future emerging epidemics and pandemics.

The membrane proximal external region (MPER) of HIV-1 gp41 is targeted by several neutralizing antibodies (NAbs) and is of interest for vaccine design. In this study, we identified novel MPER peptide mimotopes and evaluated their reactivity with HIV + plasma antibodies to characterize the diversity of the immune responses to MPER during natural infection. We utilized phage display technology to generate novel mimotopes that fit antigen-binding sites of MPER NAbs 4E10, 2F5 and Z13. Plasma antibodies from 10 HIV + patients were mapped by phage immunoprecipitation, to identify unique patient MPER binding profiles that were distinct from, and overlapping with, those of MPER NAbs. 4E10 mimotope binding profiles correlated with plasma neutralization of HIV-2/HIV-1 MPER chimeric virus, and with overall plasma neutralization breadth and potency. When administered as vaccines, 4E10 mimotopes elicited low titer NAb responses in mice. HIV mimotopes may be useful for detailed analysis of plasma antibody specificity.

Many viruses employ ATP-powered motors during assembly to translocate DNA into procapsid shells. Previous reports raise the question if motor function is modulated by substrate DNA sequence: (i) the phage T4 motor exhibits large translocation rate fluctuations and pauses and slips; (ii) evidence suggests that the phage phi29 motor contacts DNA bases during translocation; and (iii) one theoretical model, the ‘B-A scrunchworm’, predicts that ‘A-philic’ sequences that transition more easily to A-form would alter motor function. Here, we use single-molecule optical tweezers measurements to compare translocation of phage, plasmid, and synthetic A-philic, GC rich sequences by the T4 motor. We observed no significant differences in motor velocities, even with A-philic sequences predicted to show higher translocation rate at high applied force. We also observed no significant changes in motor pausing and only modest changes in slipping. To more generally test for sequence dependence, we conducted correlation analyses across pairs of packaging events. No significant correlations in packaging rate, pausing or slipping versus sequence position were detected across repeated measurements with several different DNA sequences. These studies suggest that viral genome packaging is insensitive to DNA sequence and fluctuations in packaging motor velocity, pausing and slipping are primarily stochastic temporal events.

Large biological structures are assembled from smaller, often symmetric, sub-structures. However, asymmetry among sub-structures is fundamentally important for biological function. An extreme form of asymmetry, a 12-fold-symmetric dodecameric portal complex inserted into a 5-fold-symmetric capsid vertex, is found in numerous icosahedral viruses, including tailed bacteriophages, herpesviruses, and archaeal viruses. This vertex is critical for driving capsid assembly, DNA packaging, tail attachment, and genome ejection. Here, we report the near-atomic in situ structure of the symmetry-mismatched portal vertex from bacteriophage T4. Remarkably, the local structure of portal morphs to compensate for symmetry-mismatch, forming similar interactions in different capsid environments while maintaining strict symmetry in the rest of the structure. This creates a unique and unusually dynamic symmetry-mismatched vertex that is central to building an infectious virion., In icosahedral viruses, a symmetry-mismatched portal vertex is assembled by inserting a 12-fold-symmetric portal complex into a 5-fold-symmetric capsid environment. Here, the authors report a near-atomic-resolution in situ cryo-electron microscopy structure of this symmetrically mismatched viral vertex from bacteriophage T4.

Bacteriophage (phage) T4 has served as an extraordinary model to elucidate biological structures and mechanisms. Recent discoveries on the T4 head (capsid) structure, portal vertex, and genome packaging add a significant body of new literature to phage biology. Head structures in unexpanded and expanded conformations show dramatic domain movements, structural remodeling, and a ~70% increase in inner volume while creating high-affinity binding sites for the outer decoration proteins Soc and Hoc. Small changes in intercapsomer interactions modulate angles between capsomer planes, leading to profound alterations in head length. The in situ cryo-EM structure of the symmetry-mismatched portal vertex shows the remarkable structural morphing of local regions of the portal protein, allowing similar interactions with the capsid protein in different structural environments. Conformational changes in these interactions trigger the structural remodeling of capsid protein subunits surrounding the portal vertex, which propagate as a wave of expansion throughout the capsid. A second symmetry mismatch is created when a pentameric packaging motor assembles at the outer “clip” domains of the dodecameric portal vertex. The single-molecule dynamics of the packaging machine suggests a continuous burst mechanism in which the motor subunits adjusted to the shape of the DNA fire ATP hydrolysis, generating speeds as high as 2000 bp/s.

Bacteriophage T4 has enormous potential for biomedical applications due to its large size, capsid architecture, and high payload capability for protein and DNA delivery. However, it is not very easy to genetically engineer its genome heavily modified by cytosine hydroxymethylation and glucosylation. The glucosyl hydroxymethyl cytosine (ghmC) genome of phage is completely resistant to most restriction endonucleases and exhibits various degrees of resistance to CRISPR-Cas systems. Here, we found that the type V CRISPR-Cas12a system, which shows efficient cleavage of ghmC-modified genome when compared to the type II CRISPR-Cas9 system, can be synergistically employed to generate recombinant T4 phages. Focused on surface display, we analyzed the ability of phage T4 outer capsid proteins Hoc (highly antigenic outer capsid protein) and Soc (small outer capsid protein) to tether

A “universal” vaccine design platform that can rapidly generate multiplex vaccine candidates is critically needed to control future pandemics. Here, using SARS-CoV-2 pandemic virus as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates were engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include: expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers is found to be the most potent vaccine candidate in mouse and rabbit models. Without any adjuvant, this vaccine stimulated robust immune responses, both TH1 and TH2 IgG subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new type of nanovaccine design framework might allow rapid deployment of effective phage-based vaccines against any emerging pathogen in the future.

Nucleoid Associated Proteins (NAPs) organize the bacterial chromosome within the nucleoid. The interaction of the NAP H-NS with DNA also represses specific host and xenogeneic genes. Previously, we showed that the bacteriophage T4 early protein MotB binds to DNA, co-purifies with H-NS/DNA, and improves phage fitness. Here we demonstrate using atomic force microscopy that MotB compacts the DNA with multiple MotB proteins at the center of the complex. These complexes differ from those observed with H-NS and other NAPs, but resemble those formed by the NAP-like proteins CbpA/Dps and yeast condensin. Fluorescent microscopy indicates that expression of motB in vivo, at levels like that during T4 infection, yields a significantly compacted nucleoid containing MotB and H-NS. motB overexpression dysregulates hundreds of host genes; ∼70% are within the hns regulon. In infected cells overexpressing motB, 33 T4 late genes are expressed early, and the T4 early gene repEB, involved in replication initiation, is up ∼5-fold. We postulate that MotB represents a phage-encoded NAP that aids infection in a previously unrecognized way. We speculate that MotB-induced compaction may generate more room for T4 replication/assembly and/or leads to beneficial global changes in host gene expression, including derepression of much of the hns regulon.

Developing influenza vaccines that protect against a broad range of viruses is a public health priority, and several conserved viral proteins or domains have been identified as promising targets for such vaccine development. However, none of the targets is immunogenic, and vaccine platforms that can incorporate multiple antigens with enhanced immunogenicity are desperately needed. In this study, we provided proof-of-concept for the development of next-generation influenza vaccine using T4 phage virus-like particle (VLP) platform. With extracellular domain of influenza matrix protein 2 (M2e) as a readout, we showed that more than 1,280 M2e molecules can be assembled on a 120×90 nanometer phage capsid to form T4-M2e VLPs, which are highly immunogenic and induced complete protection against influenza virus challenge without any addition adjuvant. Potentially, additional conserved antigens or molecular adjuvants could be incorporated into the T4-M2e VLPs to customize influenza vaccines to address different issues. All the components of T4 VLP vaccines can be mass-produced in E. coli in a short time, therefore, providing a rapid approach to deal with the potential influenza pandemic.

The latent HIV-1 reservoir containing stably integrated and transcriptionally silent proviruses in CD4+ T cells is a major barrier for virus eradication. Targeted reactivation of the latent reservoir remains a major challenge in establishing a path for an HIV-1 cure. Here, we investigated the possibility of reactivating the HIV-1 reservoir by targeting engineered bacteriophage T4 capsid nanoparticles to reservoir cells. The surface lattice of the 120 x 86 nm phage capsid was arrayed with CD4 binding ligands such as recombinant CD4DARPin or the HIV-1 gp140 envelope protein. When exposed to either PBMCs or the resting CD4+ T cellsin vitro, these nanoparticles caused T cells activation without inducing global T cell activation. Furthermore, the nanoparticles reactivated HIV-1 proviral transcription that led to virus assembly and release in the J-Lat cells, a cell line model of HIV-1 latency. Intriguingly, the observed T cell activation and HIV-1 latency reversal did not occur through the classic PKC or NFAT pathways suggesting the involvement of a yet unknown pathway. These studies demonstrate that engineered non-infectious bacteriophages could be potentially exploited for HIV-1 cure and other targeted T cell therapies.

Bacteria and bacteriophages (phages) have evolved potent defense and counterdefense mechanisms that allowed their survival and greatest abundance on Earth. CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) is a bacterial defense system that inactivates the invading phage genome by introducing double-strand breaks at targeted sequences. While the mechanisms of CRISPR defense have been extensively investigated, the counterdefense mechanisms employed by phages are poorly understood. Here, we report a novel counterdefense mechanism by which phage T4 restores the genomes broken by CRISPR cleavages. Catalyzed by the phage-encoded recombinase UvsX, this mechanism pairs very short stretches of sequence identity (minihomology sites), as few as 3 or 4 nucleotides in the flanking regions of the cleaved site, allowing replication, repair, and stitching of genomic fragments. Consequently, a series of deletions are created at the targeted site, making the progeny genomes completely resistant to CRISPR attack. Our results demonstrate that this is a general mechanism operating against both type II (Cas9) and type V (Cas12a) CRISPR-Cas systems. These studies uncovered a new type of counterdefense mechanism evolved by T4 phage where subtle functional tuning of preexisting DNA metabolism leads to profound impact on phage survival. IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria and use them as replication factories to assemble progeny phages. Bacteria have evolved powerful defense mechanisms to destroy the invading phages by severing their genomes soon after entry into cells. We discovered a counterdefense mechanism evolved by phage T4 to stitch back the broken genomes and restore viral infection. In this process, a small amount of genetic material is deleted or another mutation is introduced, making the phage resistant to future bacterial attack. The mutant virus might also gain survival advantages against other restriction conditions or DNA damaging events. Thus, bacterial attack not only triggers counterdefenses but also provides opportunities to generate more fit phages. Such defense and counterdefense mechanisms over the millennia led to the extraordinary diversity and the greatest abundance of bacteriophages on Earth. Understanding these mechanisms will open new avenues for engineering recombinant phages for biomedical applications.

Subunit vaccines containing one or more target antigens from pathogenic organisms represent safer alternatives to whole pathogen vaccines. However, the antigens by themselves are not sufficiently immunogenic and require additives known as adjuvants to enhance immunogenicity and protective efficacy. Assembly of the antigens into virus-like nanoparticles (VLPs) is a better approach as it allows presentation of the epitopes in a more native context. The repetitive, symmetrical, and high density display of antigens on the VLPs mimic pathogen-associated molecular patterns seen on bacteria and viruses. The antigens, thus, might be better presented to stimulate host’s innate as well as adaptive immune systems thereby eliciting both humoral and cellular immune responses. Bacteriophages such as phage T4 provide excellent platforms to generate the nanoparticle vaccines. The T4 capsid containing two non-essential outer proteins Soc and Hoc allow high density array of antigen epitopes in the form of peptides, domains, full-length proteins, or even multi-subunit complexes. Co-delivery of DNAs, targeting molecules, and/or molecular adjuvants provides additional advantages. Recent studies demonstrate that the phage T4 VLPs are highly immunogenic, do not need an adjuvant, and provide complete protection against bacterial and viral pathogens. Thus, phage T4 could potentially be developed as a “universal” VLP platform to design future multivalent vaccines against complex and emerging pathogens.

The envelope protein of human immunodeficiency virus-1 (HIV-1) and its fusion peptide are essential for cell entry and vaccine design. Here, we describe the 3.9-Å resolution structure of an envelope protein trimer from a very early transmitted founder virus (CRF01_AE T/F100) complexed with Fab from the broadly neutralizing antibody (bNAb) 8ANC195. The overall T/F100 trimer structure is similar to other reported “closed” state prefusion trimer structures. In contrast, the fusion peptide, which is exposed to solvent in reported closed structures, is sequestered (buried) in the hydrophobic core of the T/F100 trimer. A buried conformation has previously been observed in “open” state structures formed after CD4 receptor binding. The T/F100 trimer binds poorly to bNAbs including the fusion peptide-specific bNAbs PGT151 and VRC34.01. The T/F100 structure might represent a prefusion state, intermediate between the closed and open states. These observations are relevant to mechanisms of HIV-1 transmission and vaccine design., Here, the authors present the structure of the HIV envelope (Env) protein from a transmitted founder virus and show that, while the overall structure of the Env trimer is similar to other closed trimers, the fusion peptide is buried in the hydrophobic core of the trimer, which is similar to open state trimers.

Multi-subunit ring-ATPases carry out a myriad of biological functions, including genome packaging in viruses. Though the basic structures and functions of these motors have been well-established, the mechanisms of ATPase firing and motor coordination are poorly understood. Here, by direct counting using single-molecule fluorescence, we have determined that the active bacteriophage T4 DNA packaging motor consists of five subunits of gp17. By systematically doping motors with an ATPase-defective subunit and selecting single motors containing a precise count of active/inactive subunit(s), we found, unexpectedly, that the packaging motor can tolerate an inactive sub-unit. However, motors containing an inactive subunit(s) exhibit fewer DNA engagements, a higher failure rate in encapsidation, reduced packaging velocity, and increased pausing. These findings suggest a new packaging model in which the motor, by re-adjusting its grip on DNA, can skip an inactive subunit and resume DNA translocation, contrary to the prevailing notion of strict coordination amongst motor subunits of other packaging motors.

The interplay between defense and counterdefense systems of bacteria and bacteriophages has been driving the evolution of both organisms, leading to their great genetic diversity. Restriction-modification systems are well-studied defense mechanisms of bacteria, while phages have evolved covalent modifications as a counterdefense mechanism to protect their genomes against restriction. Here, we present evidence that these genome modifications might also have been selected to counter, broadly, the CRISPR-Cas systems, an adaptive bacterial defense mechanism. We found that the phage T4 genome modified by cytosine hydroxymethylation and glucosylation (ghmC) exhibits various degrees of resistance to the type V CRISPR-Cas12a system, producing orders of magnitude more progeny than the T4(C) mutant, which contains unmodified cytosines. Furthermore, the progeny accumulated CRISPR escape mutations, allowing rapid evolution of mutant phages under CRISPR pressure. A synergistic effect on phage restriction was observed when two CRISPR-Cas12a complexes were targeted to independent sites on the phage genome, another potential countermechanism by bacteria to more effectively defend themselves against modified phages. These studies suggest that the defense-counterdefense mechanisms exhibited by bacteria and phages, while affording protection against one another, also provide evolutionary benefits for both. IMPORTANCE Restriction-modification (R-M) and CRISPR-Cas systems are two well-known defense mechanisms of bacteria. Both recognize and cleave phage DNA at specific sites while protecting their own genomes. It is well accepted that T4 and other phages have evolved counterdefense mechanisms to protect their genomes from R-M cleavage by covalent modifications, such as the hydroxymethylation and glucosylation of cytosine. However, it is unclear whether such genome modifications also provide broad protection against the CRISPR-Cas systems. Our results suggest that genome modifications indeed afford resistance against CRISPR systems. However, the resistance is not complete, and it is also variable, allowing rapid evolution of mutant phages that escape CRISPR pressure. Bacteria in turn could target more than one site on the phage genome to more effectively restrict the infection of ghmC-modified phage. Such defense-counterdefense strategies seem to confer survival advantages to both the organisms, one of the possible reasons for their great diversity.

Combination antiretroviral therapy (cART) controls but does not eradicate HIV infection, HIV persistence is the principal obstacle to curing infections. The proportion of defective proviruses increases during cART, but the dynamics of this process are not well understood, and a quantitative analysis of how the proviral landscape is reshaped after cART is initiated is critical to understanding how HIV persists. Here, we studied longitudinal samples from HIV infected individuals undergoing long term cART using multiplexed Droplet Digital PCR (ddPCR) approaches to quantify the proportion of deleted proviruses in lymphocytes. In most individuals undergoing cART, HIV proviruses that contain gag are lost more quickly than those that lack gag. Increases in the fraction of gag-deleted proviruses occurred only after 1&ndash, 2 years of therapy, suggesting that the immune system, and/or toxicity of viral re-activation helps to gradually shape the proviral landscape. After 10&ndash, 15 years on therapy, there were as many as 3.5&ndash, 5 times more proviruses in which gag was deleted or highly defective than those containing intact gag. We developed a provirus-specific ddPCR approach to quantify individual clones. Investigation of a clone of cells containing a deleted HIV provirus integrated in the HORMAD2 gene revealed that the cells underwent a massive expansion shortly after cART was initiated until the clone, which was primarily in effector memory cells, dominated the population of proviruses for over 6 years. The expansion of this HIV-infected clone had substantial effects on the overall proviral population.

A viral vector that can safely and efficiently deliver large and diverse molecular cargos into cells is the holy grail of curing many human diseases. Adeno-associated virus (AAV) has been extensively used but has a very small capacity. The prokaryotic virus T4 has a large capacity but lacks natural mechanisms to enter mammalian cells. Here, we created a hybrid vector by combining T4 and AAV into one nanoparticle that possesses the advantages of both. The small 25 nm AAV particles are attached to the large 120 nm x 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc (small outer capsid protein) and Hoc (highly antigenic outer capsid protein). AAV thus "piggy-backed" on T4 capsid, by virtue of its natural ability to enter many types of human cells efficiently acts as a "driver" to deliver large cargos associated with the T4 head. This unique T4-AAV hybrid vector approach could pave the way for the development of novel therapeutics in the future.

Development of vaccines capable of eliciting broadly neutralizing antibodies (bNAbs) is a key goal to controlling the global AIDS epidemic. To be effective, bNAbs must block the capture of HIV-1 to prevent viral acquisition and establishment of reservoirs. However, the role of bNAbs, particularly during initial exposure of primary viruses to host cells, has not been fully examined. Using a sensitive, quantitative, and high-throughput qRT-PCR assay, we found that primary viruses were captured by host cells and converted into a trypsin-resistant form in less than five minutes. We discovered, unexpectedly, that bNAbs did not block primary virus capture, although they inhibited the capture of pseudoviruses/IMCs and production of progeny viruses at 48 hours. Further, viruses escaped bNAb inhibition unless the bNAbs were present in the initial minutes of exposure of virus to host cells. These findings will have important implications for HIV-1 vaccine design and determination of vaccine efficacy.

Tailed bacteriophages (phages) are one of the most abundant life forms on Earth. They encode highly efficient molecular machines to infect bacteria, but the initial interactions between a phage and a bacterium that then lead to irreversible virus attachment and infection are poorly understood. This information is critically needed to engineer machines with novel host specificities in order to combat antibiotic resistance, a major threat to global health today. The tailed phage T4 encodes a specialized device for this purpose, the long tail fiber (LTF), which allows the virus to move on the bacterial surface and find a suitable site for infection. Consequently, the infection efficiency of phage T4 is one of the highest, reaching the theoretical value of 1. Although the atomic structure of the tip of the LTF has been determined, its functional architecture and how interactions with two structurally very different Escherichia coli receptor molecules, lipopolysaccharide (LPS) and outer membrane protein C (OmpC), contribute to virus movement remained unknown. Here, by developing direct receptor binding assays, extensive mutational and biochemical analyses, and structural modeling, we discovered that the ball-shaped tip of the LTF, a trimer of gene product 37, consists of three sets of symmetrically alternating binding sites for LPS and/or OmpC. Our studies implicate reversible and dynamic interactions between these sites and the receptors. We speculate that the LTF might function as a “molecular pivot” allowing the virus to “walk” on the bacterium by adjusting the angle or position of interaction of the six LTFs attached to the six-fold symmetric baseplate.

Tailed bacteriophages (phages) are one of the most abundant life forms on Earth. They encode highly efficient molecular machines to infect bacteria, but the initial interactions between a phage and a bacterium that then lead to irreversible virus attachment and infection are poorly understood. This information is critically needed to engineer machines with novel host specificities in order to combat antibiotic resistance, a major threat to global health today. The tailed phage T4 encodes a specialized device for this purpose, the long tail fiber (LTF), which allows the virus to move on the bacterial surface and find a suitable site for infection. Consequently, the infection efficiency of phage T4 is one of the highest, reaching the theoretical value of 1. Although the atomic structure of the tip of the LTF has been determined, its functional architecture and how interactions with two structurally very different Escherichia coli receptor molecules, lipopolysaccharide (LPS) and outer membrane protein C (OmpC), contribute to virus movement remained unknown. Here, by developing direct receptor binding assays, extensive mutational and biochemical analyses, and structural modeling, we discovered that the ball-shaped tip of the LTF, a trimer of gene product 37, consists of three sets of symmetrically alternating binding sites for LPS and/or OmpC. Our studies implicate reversible and dynamic interactions between these sites and the receptors. We speculate that the LTF might function as a “molecular pivot” allowing the virus to “walk” on the bacterium by adjusting the angle or position of interaction of the six LTFs attached to the six-fold symmetric baseplate.

Tailed bacteriophages (phages) are one of the most abundant life forms on Earth. They encode highly efficient molecular machines to infect bacteria, but the initial interactions between a phage and a bacterium that then lead to irreversible virus attachment and infection are poorly understood. This information is critically needed to engineer machines with novel host specificities in order to combat antibiotic resistance, a major threat to global health today. The tailed phage T4 encodes a specialized device for this purpose, the long tail fiber (LTF), which allows the virus to move on the bacterial surface and find a suitable site for infection. Consequently, the infection efficiency of phage T4 is one of the highest, reaching the theoretical value of 1. Although the atomic structure of the tip of the LTF has been determined, its functional architecture and how interactions with two structurally very different Escherichia coli receptor molecules, lipopolysaccharide (LPS) and outer membrane protein C (OmpC), contribute to virus movement remained unknown. Here, by developing direct receptor binding assays, extensive mutational and biochemical analyses, and structural modeling, we discovered that the ball-shaped tip of the LTF, a trimer of gene product 37, consists of three sets of symmetrically alternating binding sites for LPS and/or OmpC. Our studies implicate reversible and dynamic interactions between these sites and the receptors. We speculate that the LTF might function as a “molecular pivot” allowing the virus to “walk” on the bacterium by adjusting the angle or position of interaction of the six LTFs attached to the six-fold symmetric baseplate.

An engineered viral vector of bacteriophage T4 linked to AAV delivers large payloads of genes and proteins into human cells., Development of safe and efficient nanoscale vehicles that can deliver large molecular cargos into human cells could transform future human therapies and personalized medicine. Here, we design a hybrid viral vector composed of a prokaryotic virus (bacteriophage T4) and a eukaryotic virus [adeno-associated virus (AAV)]. The small 25-nm AAV is attached to the large 120 nm × 86 nm T4 head through avidin-biotin cross-bridges using the phage decoration proteins Soc and Hoc. AAV “piggy-backed” on T4 capsid, by virtue of its natural ability to enter human cells acted as an efficient “driver,” delivering the largest payloads of foreign DNA (up to 170 kb) and protein (up to 1025 molecules) reported to date, and elicited robust immune responses in mice against flu and plague pathogens and conferred complete protection against lethal pneumonic plague challenge. The T4-AAV represents a unique platform for assembly of natural building blocks into potential therapeutics against genetic and infectious diseases.

In the original version of our article [...]

Reduced risk of HIV-1 infection correlated with antibody responses to the envelope variable 1 and 2 regions in the RV144 vaccine trial. To understand the relationship between antibody responses, V2 sequence, and structure, plasma samples (n = 16) from an early acute HIV-1 infection cohort from Thailand infected with CRF01_AE strain were analyzed for binding to V2 peptides by surface plasmon resonance. Five participants with a range of V2 binding responses at week 24 post-infection were further analyzed against a set of four overlapping V2 peptides that were designed based on envelope single-genome amplification. Antibody responses that were relatively consistent over the four segments of the V2 region or a focused response to the C-strand (residues 165–186) of the V2 region were observed. Viral escape in the V2 region resulted in significantly reduced antibody binding. Structural modeling indicated that the C-strand and the sites of viral variation were highly accessible in the open conformation of the HIV-1 Env trimer. V2 residues, 165–186 are preferentially targeted during acute infection. Residues 169–184 were also preferentially targeted by the protective immune response in the RV144 trial, thus emphasizing the importance of these residues for vaccine design.

Bacteriophages (phages) are the most abundant and widely distributed organisms on Earth, constituting a virtually unlimited resource to explore the development of biomedical therapies. The therapeutic use of phages to treat bacterial infections (“phage therapy”) was conceived by Felix d’Herelle nearly a century ago. However, its power has been realized only recently, largely due to the emergence of multi-antibiotic resistant bacterial pathogens. Progress in technologies, such as high-throughput sequencing, genome editing, and synthetic biology, further opened doors to explore this vast treasure trove. Here, we review some of the emerging themes on the use of phages against infectious diseases. In addition to phage therapy, phages have also been developed as vaccine platforms to deliver antigens as part of virus-like nanoparticles that can stimulate immune responses and prevent pathogen infections. Phage engineering promises to generate phage variants with unique properties for prophylactic and therapeutic applications. These approaches have created momentum to accelerate basic as well as translational phage research and potential development of therapeutics in the near future.

ATP-powered viral packaging motors are among the most powerful biomotors known. Motor subunits arranged in a ring repeatedly grip and translocate the DNA to package viral genomes into capsids. Here, we use single DNA manipulation and rapid solution exchange to quantify how nucleotide binding regulates interactions between the bacteriophage T4 motor and DNA substrate. With no nucleotides, there is virtually no gripping and rapid slipping occurs with only minimal friction resisting. In contrast, binding of an ATP analog engages nearly continuous gripping. Occasional slips occur due to dissociation of the analog from a gripping motor subunit, or force-induced rupture of grip, but multiple other analog-bound subunits exert high friction that limits slipping. ADP induces comparably infrequent gripping and variable friction. Independent of nucleotides, slipping arrests when the end of the DNA is about to exit the capsid. This end-clamp mechanism increases the efficiency of packaging by making it essentially irreversible., Packaging of viral DNA depends on strong molecular motors that are powered by ATP hydrolysis. Here, the authors develop a single-molecule assay to monitor how nucleotide binding regulates motor-DNA interactions and reveal a generic mechanism that prevents exit of the whole DNA from the viral capsid during packaging.

Tailed bacteriophages and herpes viruses use powerful molecular machines to package their genomes. The packaging machine consists of three components: portal, motor (large terminase; TerL) and regulator (small terminase; TerS). Portal, a dodecamer, and motor, a pentamer, form two concentric rings at the special five-fold vertex of the icosahedral capsid. Powered by ATPase, the motor ratchets DNA into the capsid through the portal channel. TerS is essential for packaging, particularly for genome recognition, but its mechanism is unknown and controversial. Structures of gear-shaped TerS rings inspired models that invoke DNA threading through the central channel. Here, we report that mutations of basic residues that line phage T4 TerS (gp16) channel do not disrupt DNA binding. Even deletion of the entire channel helix retained DNA binding and produced progeny phage in vivo. On the other hand, large oligomers of TerS (11-mers/12-mers), but not small oligomers (trimers to hexamers), bind DNA. These results suggest that TerS oligomerization creates a large outer surface, which, but not the interior of the channel, is critical for function, probably to wrap viral genome around the ring during packaging initiation. Hence, models involving TerS-mediated DNA threading may be excluded as an essential mechanism for viral genome packaging.

M-CSF increased Siglec-1 expression on macrophages, rendering them more permissive to HIV-1 infection due to interaction with V1V2 region of gp120 and associated sialic acids., Monocytes and monocyte–derived macrophages express relatively low levels of CD4. Despite this, macrophages can be effectively infected with human immunodeficiency virus type 1. Macrophages have a critical role in human immunodeficiency virus type 1 transmission; however, the mechanism or mechanisms of virus infection are poorly understood. We report that growth factors, such as granulocyte macrophage colony-stimulating factor and macrophage colony-stimulating factor affect the phenotypic profile and permissiveness of macrophages to human immunodeficiency virus type 1. Human immunodeficiency virus type 1 infection of monocyte–derived macrophages derived from granulocyte macrophage and macrophage colony-stimulating factors was predominantly facilitated by the sialic acid-binding immunoglobulin-like lectin-1. The number of sialic acid-binding immunoglobulin-like lectin receptors on macrophage colony-stimulating factor–derived monocyte–derived macrophages was significantly greater than on granulocyte macrophage colony-stimulating factor–derived monocyte–derived macrophages, and correspondingly, human immunodeficiency virus type 1 infection was greater in the macrophage colony-stimulating factor–derived monocyte–derived macrophages. Single-genome analysis and quantitative reverse transcriptase-polymerase chain reaction revealed that the differences in infectivity was not due to differences in viral fitness or in viral variants with differential infectivity but was due to reduced viral entry into the granulocyte macrophage colony-stimulating factor–derived monocyte–derived macrophages. Anti-sialic acid-binding immunoglobulin-like lectin, trimeric glycoprotein 145, and scaffolded V1V2 proteins were bound to sialic acid-binding immunoglobulin-like lectin and significantly reduced human immunodeficiency virus type 1 entry and infection. Furthermore, sialic acid residues present in the V1V2 region of the envelope protein mediated human immunodeficiency virus type 1 interaction with sialic acid-binding immunoglobulin-like lectin and entry into macrophage colony-stimulating factor–derived monocyte–derived macrophages. Removal of sialic acid residues or glycans from scaffolded V1V2 protein decreased human immunodeficiency virus type 1 infectivity. These results highlight the importance of sialic acids on the V1V2 region in binding to sialic acid-binding immunoglobulin-like lectin and suggest that the unusually long surface-exposed sialic acid-binding immunoglobulin-like lectin might aid in the capture and entry of human immunodeficiency virus type 1 into monocyte–derived macrophages.

Following the deadly anthrax attacks of 2001, the Centers for Disease Control and Prevention (CDC) determined that Bacillus anthracis and Yersinia pestis that cause anthrax and plague, respectively, are two Tier 1 select agents that pose the greatest threat to the national security of the United States. Both cause rapid death, in 3 to 6 days, of exposed individuals. We engineered a virus nanoparticle vaccine using bacteriophage T4 by incorporating key antigens of both B. anthracis and Y. pestis into one formulation. Two doses of this vaccine provided complete protection against both inhalational anthrax and pneumonic plague in animal models. This dual anthrax-plague vaccine is a strong candidate for stockpiling against a potential bioterror attack involving either one or both of these biothreat agents. Further, our results establish the T4 nanoparticle as a novel platform to develop multivalent vaccines against pathogens of high public health significance., Bacillus anthracis and Yersinia pestis, the causative agents of anthrax and plague, respectively, are two of the deadliest pathogenic bacteria that have been used as biological warfare agents. Although Biothrax is a licensed vaccine against anthrax, no Food and Drug Administration-approved vaccine exists for plague. Here, we report the development of a dual anthrax-plague nanoparticle vaccine employing bacteriophage (phage) T4 as a platform. Using an in vitro assembly system, the 120- by 86-nm heads (capsids) of phage T4 were arrayed with anthrax and plague antigens fused to the small outer capsid protein Soc (9 kDa). The antigens included the anthrax protective antigen (PA) (83 kDa) and the mutated (mut) capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system from Y. pestis (F1mutV) (56 kDa). These viral nanoparticles elicited robust anthrax- and plague-specific immune responses and provided complete protection against inhalational anthrax and/or pneumonic plague in three animal challenge models, namely, mice, rats, and rabbits. Protection was demonstrated even when the animals were simultaneously challenged with lethal doses of both anthrax lethal toxin and Y. pestis CO92 bacteria. Unlike the traditional subunit vaccines, the phage T4 vaccine uses a highly stable nanoparticle scaffold, provides multivalency, requires no adjuvant, and elicits broad T-helper 1 and 2 immune responses that are essential for complete clearance of bacteria during infection. Therefore, phage T4 is a unique nanoparticle platform to formulate multivalent vaccines against high-risk pathogens for national preparedness against potential bioterror attacks and emerging infections.

Phages show an elevated mutation rate and remarkably rapid evolution when attacked by the bacterial CRISPR/Cas system., Bacteria and bacteriophages arm themselves with various defensive and counterdefensive mechanisms to protect their own genome and degrade the other’s. CRISPR (clustered regularly interspaced short palindromic repeat)–Cas (CRISPR-associated) is an adaptive bacterial defense mechanism that recognizes short stretches of invading phage genome and destroys it by nuclease attack. Unexpectedly, we discovered that the CRISPR-Cas system might also accelerate phage evolution. When Escherichia coli bacteria containing CRISPR-Cas9 were infected with phage T4, its cytosine hydroxymethylated and glucosylated genome was cleaved poorly by Cas9 nuclease, but the continuing CRISPR-Cas9 pressure led to rapid evolution of mutants that accumulated even by the time a single plaque was formed. The mutation frequencies are, remarkably, approximately six orders of magnitude higher than the spontaneous mutation frequency in the absence of CRISPR pressure. Our findings lead to the hypothesis that the CRISPR-Cas might be a double-edged sword, providing survival advantages to both bacteria and phages, leading to their coevolution and abundance on Earth.

Translocation of viral double-stranded DNA (dsDNA) into the icosahedral prohead shell is catalyzed by TerL, a motor protein that has ATPase, endonuclease, and translocase activities. TerL, following endonucleolytic cleavage of immature viral DNA concatemer recognized by TerS, assembles into a pentameric ring motor on the prohead's portal vertex and uses ATP hydrolysis energy for DNA translocation. TerL's N-terminal ATPase is connected by a hinge to the C-terminal endonuclease. Inchworm models propose that modest domain motions accompanying ATP hydrolysis are amplified, through changes in electrostatic interactions, into larger movements of the C-terminal domain bound to DNA. In phage ϕ29, four of the five TerL subunits sequentially hydrolyze ATP, each powering translocation of 2.5 bp. After one viral genome is encapsidated, the internal pressure signals termination of packaging and ejection of the motor. Current focus is on the structures of packaging complexes and the dynamics of TerL during DNA packaging, endonuclease regulation, and motor mechanics.

Background: HIV-1 envelope trimer is a candidate for designing an effective HIV vaccine. Results: gp140 attached to Strep-tag through a long linker is used to purify HIV trimers. Cleaved, uncleaved, and fully and partially glycosylated trimers are characterized. Conclusion: Cleaved and glycosylated gp140 assembles into authentic propeller-shaped trimers. Significance: This system could generate HIV-1 trimers for clinical trials and vaccine manufacture., The trimeric envelope spike of HIV-1 mediates virus entry into human cells. The exposed part of the trimer, gp140, consists of two noncovalently associated subunits, gp120 and gp41 ectodomain. A recombinant vaccine that mimics the native trimer might elicit entry-blocking antibodies and prevent virus infection. However, preparation of authentic HIV-1 trimers has been challenging. Recently, an affinity column containing the broadly neutralizing antibody 2G12 has been used to capture recombinant gp140 and prepare trimers from clade A BG505 that naturally produces stable trimers. However, this antibody-based approach may not be as effective for the diverse HIV-1 strains with different epitope signatures. Here, we report a new and simple approach to produce HIV-1 envelope trimers. The C terminus of gp140 was attached to Strep-tag II with a long linker separating the tag from the massive trimer base and glycan shield. This allowed capture of nearly homogeneous gp140 directly from the culture medium. Cleaved, uncleaved, and fully or partially glycosylated trimers from different clade viruses were produced. Extensive biochemical characterizations showed that cleavage of gp140 was not essential for trimerization, but it triggered a conformational change that channels trimers into correct glycosylation pathways, generating compact three-blade propeller-shaped trimers. Uncleaved trimers entered aberrant pathways, resulting in hyperglycosylation, nonspecific cross-linking, and conformational heterogeneity. Even the cleaved trimers showed microheterogeneity in gp41 glycosylation. These studies established a broadly applicable HIV-1 trimer production system as well as generating new insights into their assembly and maturation that collectively bear on the HIV-1 vaccine design.

The 3.3-A cryo-EM structure of the 860-A-diameter isometric mutant bacteriophage T4 capsid has been determined. WT T4 has a prolate capsid characterized by triangulation numbers (T numbers) Tend = 13 for end caps and Tmid = 20 for midsection. A mutation in the major capsid protein, gp23, produced T=13 icosahedral capsids. The capsid is stabilized by 660 copies of the outer capsid protein, Soc, which clamp adjacent gp23 hexamers. The occupancies of Soc molecules are proportional to the size of the angle between the planes of adjacent hexameric capsomers. The angle between adjacent hexameric capsomers is greatest around the fivefold vertices, where there is the largest deviation from a planar hexagonal array. Thus, the Soc molecules reinforce the structure where there is the greatest strain in the gp23 hexagonal lattice. Mutations that change the angles between adjacent capsomers affect the positions of the pentameric vertices, resulting in different triangulation numbers in bacteriophage T4. The analysis of the T4 mutant head assembly gives guidance to how other icosahedral viruses reproducibly assemble into capsids with a predetermined T number, although the influence of scaffolding proteins is also important.

Bacteriophages likely constitute the largest biomass on Earth. However, very few phage genomes have been well-characterized, the tailed phage T4 genome being one of them. Even in T4, much of the genome remained uncharacterized. The classical genetic strategies are tedious, compounded by genome modifications such as cytosine hydroxylmethylation and glucosylation which makes T4 DNA resistant to most restriction endonucleases. Here, using the type-II CRISPR-Cas9 system, we report the editing of both modified (ghm-Cytosine) and unmodified (Cytosine) T4 genomes. The modified genome, however, is less susceptible to Cas9 nuclease attack when compared to the unmodified genome. The efficiency of restriction of modified phage infection varied greatly in a spacer-dependent manner, which explains some of the previous contradictory results. We developed a genome editing strategy by codelivering into E. coli a CRISPR-Cas9 plasmid and a donor plasmid containing the desired mutation(s). Single and multiple point mutations, insertions and deletions were introduced into both modified and unmodified genomes. As short as 50-bp homologous flanking arms were sufficient to generate recombinants that can be selected under the pressure of CRISPR-Cas9 nuclease. A 294-bp deletion in RNA ligase gene rnlB produced viable plaques, demonstrating the usefulness of this editing strategy to determine the essentiality of a given gene. These results provide the first demonstration of phage T4 genome editing that might be extended to other phage genomes in nature to create useful recombinants for phage therapy applications.

The speed at which a molecular motor operates is critically important for the survival of a virus or an organism but very little is known about the underlying mechanisms. Tailed bacteriophage T4 employs one of the fastest and most powerful packaging motors, a pentamer of gp17 that translocates DNA at a rate of up to ∼2000-bp/s. We hypothesize, guided by structural and genetic analyses, that a unique hydrophobic environment in the catalytic space of gp17-adenosine triphosphatase (ATPase) determines the rate at which the ‘lytic water’ molecule is activated and OH− nucleophile is generated, in turn determining the speed of the motor. We tested this hypothesis by identifying two hydrophobic amino acids, M195 and F259, in the catalytic space of gp17-ATPase that are in a position to modulate motor speed. Combinatorial mutagenesis demonstrated that hydrophobic substitutions were tolerated but polar or charged substitutions resulted in null or cold-sensitive/small-plaque phenotypes. Quantitative biochemical and single-molecule analyses showed that the mutant motors exhibited 1.8- to 2.5-fold lower rate of ATP hydrolysis, 2.5- to 4.5-fold lower DNA packaging velocity, and required an activator protein, gp16 for rapid firing of ATPases. These studies uncover a speed control mechanism that might allow selection of motors with optimal performance for organisms’ survival.