Structural basis for DNA recognition by a viral genome-packaging machine.

DNA recognition is critical for assembly of double-stranded DNA viruses, particularly for the initiation of packaging the viral genome into the capsid. The key component that recognizes viral DNA is the small terminase protein. Despite prior studies, the molecular mechanism for DNA recognition remained elusive. Here, we address this question by identifying the minimal site in the bacteriophage HK97 genome specifically recognized by the small terminase and determining the structure of this complex by cryoEM. The circular small terminase employs an entirely unexpected mechanism in which DNA transits through the central tunnel, and sequence-specific recognition takes place as it emerges. This recognition stems from a substructure formed by the N- and C-terminal segments of two adjacent protomers which are unstructured when DNA is absent. Such interaction ensures continuous engagement of the small terminase with DNA, enabling it to slide along the DNA while simultaneously monitoring its sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the specific cos sequence.

Methodological approaches to studying phase separation and HIV-1 replication: Current and future perspectives.

This article reviews tried-and-tested methodologies that have been employed in the first studies on phase separating properties of structural, RNA-binding and catalytic proteins of HIV-1. These are described here to stimulate interest for any who may want to initiate similar studies on virus-mediated liquid-liquid phase separation. Such studies serve to better understand the life cycle and pathogenesis of viruses and open the door to new therapeutics.

The assembly-defective phenotype of an FIV Gag polyprotein containing the SIV nucleocapsid zinc finger motifs is reversed by including the SIV SP2 domain in the chimeric protein.

To gain insight into the functional relationship between the nucleocapsid (NC) domains of the Gag polyproteins of feline and simian immunodeficiency viruses, FIV and SIV, respectively, we generated two FIV Gag chimeric proteins containing different SIV NC and gag sequences. A chimeric FIV Gag protein (NC1) containing the SIV two zinc fingers motifs was incapable of assembling into virus-like particles. By contrast, another Gag chimera (NC2) differing from NC1 by the replacement of the C-terminal region of the FIV NC with SIV SP2 produced particles as efficiently as wild-type FIV Gag. Of note, when the chimeric NC2 Gag polyprotein was expressed in the context of the proviral DNA in feline CrFK cells, wild-type levels of virions were produced which encapsidated 50% of genomic RNA when compared to the wild-type virus.

Arg18 Substitutions Reveal the Capacity of the HIV-1 Capsid Protein for Non-Fullerene Assembly.

In the fullerene cone HIV-1 capsid, the central channels of the hexameric and pentameric capsomers each contain a ring of arginine (Arg18) residues that perform essential roles in capsid assembly and function. In both the hexamer and pentamer, the Arg18 rings coordinate inositol hexakisphosphate, an assembly and stability factor for the capsid. Previously, it was shown that amino-acid substitutions of Arg18 can promote pentamer incorporation into capsid-like particles (CLPs) that spontaneously assemble in vitro under high-salt conditions. Here, we show that these Arg18 mutant CLPs contain a non-canonical pentamer conformation and distinct lattice characteristics that do not follow the fullerene geometry of retroviral capsids. The Arg18 mutant pentamers resemble the hexamer in intra-oligomeric contacts and form a unique tetramer-of-pentamers that allows for incorporation of an octahedral vertex with a cross-shaped opening in the hexagonal capsid lattice. Our findings highlight an unexpected degree of structural plasticity in HIV-1 capsid assembly.

The SARS-CoV-2 nucleoprotein associates with anionic lipid membranes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N), and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we used several lipid binding assays and found the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, we show lipid binding occurs in the N protein C-terminal domain, which is supported by extensive in silico analysis. We demonstrate anionic lipid binding occurs for both the free and the N oligomeric forms, suggesting N can associate with membranes in the nucleocapsid form. Based on these results, we present a lipid-dependent model based on in vitro, cellular, and in silico data for the recruitment of N to assembly sites in the lifecycle of SARS-CoV-2.

Single-Virion Analysis: A Method to Visualize HIV-1 Particle Content Using Fluorescence Microscopy.

HIV-1 virions incorporate viral RNA, cellular RNAs, and proteins during the assembly process. Some of these components, such as the viral RNA genome and viral proteins, are essential for viral replication, whereas others, such as host innate immune proteins, can inhibit virus replication. Therefore, analyzing the virion content is an integral part of studying HIV-1 replication. Traditionally, virion contents have been examined using biochemical assays, which can provide information on the presence or absence of the molecule of interest but not its distribution in the virion population. Here, we describe a method, single-virion analysis, that directly examines the presence of molecules of interest in individual viral particles using fluorescence microscopy. Thus, this method can detect both the presence and the distribution of molecules of interest in the virion population. Single-virion analysis was first developed to study HIV-1 RNA genome packaging. In this assay, HIV-1 unspliced RNA is labeled with a fluorescently tagged RNA-binding protein (protein A) and some of the Gag proteins are labeled with a different fluorescent protein (protein B). Using fluorescence microscopy, HIV-1 particles can be identified by the fluorescent protein B signal and the presence of unspliced HIV-1 RNA can be identified by the fluorescent protein A signal. Therefore, the proportions of particles that contain unspliced RNA can be determined by the fraction of Gag particles that also have a colocalized RNA signal. By tagging the molecule of interest with fluorescent proteins, single-virion analysis can be easily adapted to study the incorporation of other viral or host cell molecules into particles. Indeed, this method has been adapted to examine the proportion of HIV-1 particles that contain APOBEC3 proteins and the fraction of particles that contain a modified Gag protein. Therefore, single-virion analysis is a flexible method to study the nucleic acid and protein content of HIV-1 particles.

Computational and experimental identification of keystone interactions in Ebola virus matrix protein VP40 dimer formation.

The Ebola virus (EBOV) is a lipid-enveloped virus with a negative sense RNA genome that can cause severe and often fatal viral hemorrhagic fever. The assembly and budding of EBOV is regulated by the matrix protein, VP40, which is a peripheral protein that associates with anionic lipids at the inner leaflet of the plasma membrane. VP40 is sufficient to form virus-like particles (VLPs) from cells, which are nearly indistinguishable from authentic virions. Due to the restrictions of studying EBOV in BSL-4 facilities, VP40 has served as a surrogate in cellular studies to examine the EBOV assembly and budding process from the host cell plasma membrane. VP40 is a dimer where inhibition of dimer formation halts budding and formation of new VLPs as well as VP40 localization to the plasma membrane inner leaflet. To better understand VP40 dimer stability and critical amino acids to VP40 dimer formation, we integrated computational approaches with experimental validation. Site saturation/alanine scanning calculation, combined with molecular mechanics-based generalized Born with Poisson-Boltzmann surface area (MM-GB/PBSA) method and molecular dynamics simulations were used to predict the energetic contribution of amino acids to VP40 dimer stability and the hydrogen bonding network across the dimer interface. These studies revealed several previously unknown interactions and critical residues predicted to impact VP40 dimer formation. In vitro and cellular studies were then pursued for a subset of VP40 mutations demonstrating reduction in dimer formation (in vitro) or plasma membrane localization (in cells). Together, the computational and experimental approaches revealed critical residues for VP40 dimer stability in an alpha-helical interface (between residues 106-117) as well as in a loop region (between residues 52-61) below this alpha-helical region. This study sheds light on the structural origins of VP40 dimer formation and may inform the design of a small molecule that can disrupt VP40 dimer stability.

Multi-Gene Recombinant Baculovirus Expression Systems: From Inception to Contemporary Applications.

Many protein expression systems are primarily utilised to produce a single, specific recombinant protein. In contrast, most biological processes such as virus assembly rely upon a complex of several interacting proteins rather than the activity of a sole protein. The high complexity of the baculovirus genome, coupled with a multiphase replication cycle incorporating distinct transcriptional steps, made it the ideal system to manipulate for high-level expression of a single, or co-expression of multiple, foreign proteins within a single cell. We have developed and utilised a series of recombinant baculovirus systems to unravel the sequential assembly process of a complex non-enveloped model virus, bluetongue virus (BTV). The high protein yields expressed by the baculovirus system not only facilitated structure-function analysis of each viral protein but were also advantageous to crystallography studies and supported the first atomic-level resolution of a recombinant viral protein, the major BTV capsid protein. Further, the formation of recombinant double-shelled virus-like particles (VLPs) provided insights into the structure-function relationships among the four major structural proteins of the BTV whilst also representing a potential candidate for a viral vaccine. The baculovirus multi-gene expression system facilitated the study of structurally complex viruses (both non-enveloped and enveloped viruses) and heralded a new generation of viral vaccines.

Nelfinavir inhibition of Kaposi's sarcoma-associated herpesvirus protein expression and capsid assembly.

BACKGROUND: Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions. METHODS: For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in greater detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication. RESULTS: We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 min. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein, ORF26, was markedly reduced. This is an essential protein required for herpesvirus capsid assembly. CONCLUSIONS: Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. This is likely because of the effect of nelfinavir on the ISR and thus protein synthesis and accumulation of the essential triplex capsid protein, ORF26. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Minor electrostatic changes robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

HIV-1 RNA genome packaging: it's G-rated.

A member of the Retroviridae, human immunodeficiency virus type 1 (HIV-1), uses the RNA genome packaged into nascent virions to transfer genetic information to its progeny. The genome packaging step is a highly regulated and extremely efficient process as a vast majority of virus particles contain two copies of full-length unspliced HIV-1 RNA that form a dimer. Thus, during virus assembly HIV-1 can identify and selectively encapsidate HIV-1 unspliced RNA from an abundant pool of cellular RNAs and various spliced HIV-1 RNAs. Several "G" features facilitate the packaging of a dimeric RNA genome. The viral polyprotein Gag orchestrates virus assembly and mediates RNA genome packaging. During this process, Gag preferentially binds unpaired guanosines within the highly structured 5' untranslated region (UTR) of HIV-1 RNA. In addition, the HIV-1 unspliced RNA provides a scaffold that promotes Gag:Gag interactions and virus assembly, thereby ensuring its packaging. Intriguingly, recent studies have shown that the use of different guanosines at the junction of U3 and R as transcription start sites results in HIV-1 unspliced RNA species with 99.9% identical sequences but dramatically distinct 5' UTR conformations. Consequently, one species of unspliced RNA is preferentially packaged over other nearly identical RNAs. These studies reveal how conformations affect the functions of HIV-1 RNA elements and the complex regulation of HIV-1 replication. In this review, we summarize cis- and trans-acting elements critical for HIV-1 RNA packaging, locations of Gag:RNA interactions that mediate genome encapsidation, and the effects of transcription start sites on the structure and packaging of HIV-1 RNA.

Minor changes in electrostatics robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine (PS) and PI(4,5)P2 in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles (VLPs). In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

Antibody inhibition of influenza A virus assembly and release.

Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal antibodies (mAbs) has been shown to restrict replication at the level of virus assembly and release, it remains unclear how potent and pervasive this mechanism of protection is, due in part to the challenge of separating this effect from other aspects of antibody function. To address this question, we developed imaging-based assays to determine how effectively a broad range of mAbs against the IAV surface proteins can specifically restrict viral egress. We find that classically neutralizing antibodies against hemagglutinin are broadly multifunctional, inhibiting virus assembly and release at concentrations 1-20-fold higher than the concentrations at which they inhibit viral entry. These antibodies are also capable of altering the morphological features of shed virions, reducing the proportion of filamentous particles. We find that antibodies against neuraminidase and M2 also restrict viral egress and that inhibition by anti-neuraminidase mAbs is only partly attributable to a loss in enzymatic activity. In all cases, antigen crosslinking-either on the surface of the infected cell, between the viral and cell membrane, or both-plays a critical role in inhibition, and we are able to distinguish between these modes experimentally and through a structure-based computational model. Together, these results provide a framework for dissecting antibody multifunctionality that could help guide the development of improved therapeutic antibodies or vaccines and that can be extended to other viral families and antibody isotypes.IMPORTANCEAntibodies against influenza A virus provide multifaceted protection against infection. Although sensitive and quantitative assays are widely used to measure inhibition of viral attachment and entry, the ability of diverse antibodies to inhibit viral egress is less clear. We address this challenge by developing an imaging-based approach to measure antibody inhibition of virus release across a panel of monoclonal antibodies targeting the influenza A virus surface proteins. Using this approach, we find that inhibition of viral egress is common and can have similar potency to the ability of an antibody to inhibit viral entry. Insights into this understudied aspect of antibody function may help guide the development of improved countermeasures.

PI(4,5)P2 binding sites in the Ebola virus matrix protein VP40 modulate assembly and budding.

Ebola virus (EBOV) causes severe hemorrhagic fever in humans and is lethal in a large percentage of those infected. The EBOV matrix protein viral protein 40 kDa (VP40) is a peripheral binding protein that forms a shell beneath the lipid bilayer in virions and virus-like particles (VLPs). VP40 is required for virus assembly and budding from the host cell plasma membrane. VP40 is a dimer that can rearrange into oligomers at the plasma membrane interface, but it is unclear how these structures form and how they are stabilized. We therefore investigated the ability of VP40 to form stable oligomers using in vitro and cellular assays. We characterized two lysine-rich regions in the VP40 C-terminal domain (CTD) that bind phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and play distinct roles in lipid binding and the assembly of the EBOV matrix layer. The extensive analysis of VP40 with and without lipids by hydrogen deuterium exchange mass spectrometry revealed that VP40 oligomers become extremely stable when VP40 binds PI(4,5)P2. The PI(4,5)P2-induced stability of VP40 dimers and oligomers is a critical factor in VP40 oligomerization and release of VLPs from the plasma membrane. The two lysine-rich regions of the VP40 CTD have different roles with respect to interactions with plasma membrane phosphatidylserine (PS) and PI(4,5)P2. CTD region 1 (Lys221, Lys224, and Lys225) interacts with PI(4,5)P2 more favorably than PS and is important for VP40 extent of oligomerization. In contrast, region 2 (Lys270, Lys274, Lys275, and Lys279) mediates VP40 oligomer stability via lipid interactions and has a more prominent role in release of VLPs.

Evidence that an interaction between the respiratory syncytial virus F and G proteins at the distal ends of virus filaments mediates efficient multiple cycle infection.

Evidence for a stable interaction between the respiratory syncytial virus (RSV) F and G proteins on the surface of virus filaments was provided using antibody immunoprecipitation studies on purified RSV particles, and by the in situ analysis on the surface of RSV-infected cells using the proximity ligation assay. Imaging of the F and G protein distribution on virus filaments suggested that this protein complex was localised at the distal ends of the virus filaments, and suggested that this protein complex played a direct role in mediating efficient localised cell-to-cell virus transmission. G protein expression was required for efficient localised cell-to-cell transmission of RSV in cell monolayers which provided evidence that this protein complex mediates efficient multiple cycle infection. Collectively, these data provide evidence that F and G proteins form a complex on the surface of RSV particles, and that a role for this protein complex in promoting virus transmission is suggested.

Defining the Assembleome of the Respiratory Syncytial Virus.

During respiratory syncytial virus (RSV) particle assembly, the mature RSV particles form as filamentous projections on the surface of RSV-infected cells. The RSV assembly process occurs at the / on the cell surface that is modified by a virus infection, involving a combination of several different host cell factors and cellular processes. This induces changes in the lipid composition and properties of these lipid microdomains, and the virus-induced activation of associated Rho GTPase signaling networks drives the remodeling of the underlying filamentous actin (F-actin) cytoskeleton network. The modified sites that form on the surface of the infected cells form the nexus point for RSV assembly, and in this review chapter, they are referred to as the RSV assembleome. This is to distinguish these unique membrane microdomains that are formed during virus infection from the corresponding membrane microdomains that are present at the cell surface prior to infection. In this article, an overview of the current understanding of the processes that drive the formation of the assembleome during RSV particle assembly is given.

Nelfinavir Inhibition of Kaposi's sarcoma-associated herpesvirus protein expression and capsid assembly.

Background: Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) infections aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions. Methods: For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication. Results: We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 minutes. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein ORF26 was markedly reduced. This is an essential protein required for herpesvirus capsid assembly. Conclusions: Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Human Mannose Receptor 1 Attenuates HIV-1 Infectivity in a Virus Isolate-Specific Manner.

Human mannose receptor 1 (hMRC1) is a transmembrane glycoprotein that belongs to the C-type lectin family and is expressed on the surface of most tissue macrophages. hMRC1 contributes to the binding and transmission of HIV-1 and is involved in the endocytic uptake of HIV-1 for subsequent antigen presentation. We previously reported that hMRC1 functions as an antiviral factor by inhibiting virus release through a BST-2-like mechanism. The inhibition of virus release was not virus isolate-specific and, surprisingly, was not Env-dependent. We now report on another hMRC1 antiviral function that affects the infectivity of viral particles. Unlike its effect on virus release, the inhibition of viral infectivity by hMRC1 was virus isolate-specific. An analysis of chimeric Env revealed that the Env V3 region was a critical determinant for the inhibitory effect of hMRC1. Of note, exogenously expressed hMRC1 was packaged into viral particles in an Env-independent manner. Co-immunoprecipitation studies revealed a strong interaction of the hMRC1-sensitive NL43 Env with hMRC1, while the hMRC1-insensitive Envs of AD8 and 49.5 isolates interacted poorly if at all with hMRC1. An analysis of a panel of Transmitted/Founder (T/F) viruses revealed that all of them were R5-tropic, and more than half of them were inhibited by hMRC1. The detailed mechanism of how hMRC1 inhibits viral infectivity remains to be investigated. However, the high-affinity binding of hMRC1 to Env may cause a conformational change around the Env V3 region or obstruct the Env V3 region and may make it inaccessible for subsequent interaction with the coreceptor during virus entry.

Structure of the Borrelia Bacteriophage φBB1 Procapsid.

Bacteriophages of Borrelia burgdorferi are a biologically important but under-investigated feature of the Lyme disease-causing spirochete. No virulent borrelial viruses have been identified, but all B. burgdorferi isolates carry a prophage φBB1 as resident circular plasmids. Like its host, the φBB1 phage is quite distinctive and shares little sequence similarity with other known bacteriophages. We expressed φBB1 head morphogenesis proteins in Escherichia coli which resulted in assembly of homogeneous prolate procapsid structures and used cryo-electron microscopy to determine the three-dimensional structure of these particles. The φBB1 procapsids consist of 415 copies of the major capsid protein and an equal combined number of three homologous capsid decoration proteins that form trimeric knobs on the outside of the particle. One of the end vertices of the particle is occupied by a portal assembled from twelve copies of the portal protein. The φBB1 scaffolding protein is entirely α-helical and has an elongated shape with a small globular domain in the middle. Within the tubular section of the procapsid, the internal scaffold is built of stacked rings, each composed of 32 scaffolding protein molecules, which run in opposite directions from both caps with a heterogeneous part in the middle. Inside the portal-containing cap, the scaffold is organized asymmetrically with ten scaffolding protein molecules bound to the portal. The φBB1 procapsid structure provides better insight into the vast structural diversity of bacteriophages and presents clues of how elongated bacteriophage particles might be assembled.