Effect of macromolecular crowding agents on human immunodeficiency virus type 1 capsid protein assembly in vitro

Previous studies on the self-assembly of capsid protein CA of human immunodeficiency virus type 1 (HIV-1) in vitro have provided important insights on the structure and assembly of the mature HIV-1 capsid. However, CA polymerization in vitro was previously observed to occur only at very high ionic strength. Here, we have analyzed the effects on CA assembly in vitro of adding unrelated, inert macromolecules (crowding agents), aimed at mimicking the crowded (very high macromolecular effective concentration) environment within the HIV-1 virion. Crowding agents induced fast and efficient polymerization of CA even at low (close to physiological) ionic strength. The hollow cylinders thus assembled were indistinguishable in shape and dimensions from those formed in dilute protein solutions at high ionic strength. However, two important differences were noted: (i) disassembly by dilution of the capsid-like particles was undetectable at very high ionic strength, but occurred rapidly at low ionic strength in the presence of a crowding agent, and (ii) a variant CA from a presumed infectious HIV-1 with mutations at the CA dimerization interface was unable to assemble at any ionic strength in the absence of a crowding agent; in contrast, this mutation allowed efficient assembly, even at low ionic strength, when a crowding agent was used. The use of a low ionic strength and inert macromolecules to mimic the crowded environment inside the HIV-1 virion may lead to a better in vitro evaluation of the effects of conditions, mutations or/and other molecules, including potential antiviral compounds, on HIV-1 capsid assembly, stability and disassembly.     

Weak protein-protein interactions are sufficient to drive assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.     

Association Equilibrium of the HIV-1 Capsid Protein in a Crowded Medium Reveals that Hexamerization during Capsid Assembly Requires a Functional C-Domain Dimerization Interface

Polymerization of the intact capsid protein (CA) of HIV-1 into mature capsidlike particles at physiological ionic strength in vitro requires macromolecularly crowded conditions that approach those inside the virion, where the mature capsid is assembled in vivo. The capsid is organized as a hexameric lattice. CA subunits in each hexamer are connected through interfaces that involve the CA N-terminal domain (NTD); pairs of CA subunits belonging to different hexamers are connected through a different interface that involves the C-terminal domain (CTD). At physiological ionic strength in noncrowded conditions, CA subunits homodimerize through this CTD-CTD interface, but do not hexamerize through the other interfaces (those involving the NTD). Here we have investigated whether macromolecular crowding conditions are able to promote hexamerization of the isolated NTD and/or full-length CA (with an inactive CTD-CTD interface to prevent polymerization). The oligomerization state of the proteins was determined using analytical ultracentrifugation in the absence or presence of high concentrations of an inert macromolecular crowding agent. Under the same conditions that promoted efficient assembly of intact CA dimers, neither NTD nor CA with an inactive CTD-CTD interface showed any tendency to form hexamers or any other oligomer. This inability to hexamerize was observed even in macromolecularly crowded conditions. The results indicate that a functional CTD-CTD interface is strictly required for hexamerization of HIV-1 CA through the other interfaces. Together with previous results, these observations suggest that establishment of NTD-CTD interactions involved in CA hexamerization during mature HIV-1 capsid assembly requires a homodimerization-dependent conformational switching of CTD.     

The HIV-1 capsid protein C-terminal domain in complex with a virus assembly inhibitor

Immature HIV particles bud from infected cells after assembly at the cytoplasmic side of cellular membranes. This assembly is driven by interactions between Gag polyproteins. Mature particles, each containing a characteristic conical core, are later generated by proteolytic maturation of Gag in the virion. The C-terminal domain of the HIV-1 capsid protein (C-CA) has been shown to contain oligomerization determinants essential for particle assembly. Here we report the 1.7-A-resolution crystal structure of C-CA in complex with a peptide capable of inhibiting immature- and mature-like particle assembly in vitro. The peptide inserts as an amphipathic alpha-helix into a conserved hydrophobic groove of C-CA, resulting in formation of a compact five-helix bundle with altered dimeric interactions. This structure thus reveals the details of an allosteric site in the HIV capsid protein that can be targeted for antiviral therapy.     

The Mason-Pfizer monkey virus internal scaffold domain enables in vitro assembly of human immunodeficiency virus type 1 Gag

The Mason-Pfizer monkey virus (M-PMV) Gag protein possesses the ability to assemble into an immature capsid when synthesized in a reticulocyte lysate translation system. In contrast, the human immunodeficiency virus (HIV) Gag protein is incapable of assembly in parallel assays. To enable the assembly of HIV Gag, we have combined or inserted regions of M-PMV Gag into HIV Gag. By both biochemical and morphological criteria, several of these chimeric Gag molecules are capable of assembly into immature capsid-like structures in this in vitro system. Chimeric species containing large regions of M-PMV Gag fused to HIV Gag sequences failed to assemble, while species consisting of only the M-PMV p12 region, and its internal scaffold domain (ISD), fused to HIV Gag were capable of assembly, albeit at reduced kinetics compared to M-PMV Gag. The ability of the ISD to induce assembly of HIV Gag, which normally assembles at the plasma membrane, suggests a common requirement for a concentrating factor in retrovirus assembly. Despite the dramatic effect of the ISD on chimera assembly, the function of HIV Gag domains in that process was found to remain essential, since an assembly-defective mutant of HIV CA, M185A, abolished assembly when introduced into the chimera. This continued requirement for HIV Gag domain function in the assembly of chimeric molecules will allow this in vitro system to be used for the analysis of potential inhibitors of HIV immature particle assembly.     

N-Terminal Extension of Human Immunodeficiency Virus Capsid Protein Converts the In Vitro Assembly Phenotype from Tubular to Spherical Particles

Expression of retroviral Gag polyproteins is sufficient for morphogenesis of virus-like particles with a spherical immature protein shell. Proteolytic cleavage of Gag into the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains (in the case of human immunodeficiency virus [HIV]) leads to condensation to the mature cone-shaped core. We have analyzed the formation of spherical or cylindrical particles on in vitro assembly of purified HIV proteins or inside Escherichia coli cells. CA protein alone yielded cylindrical particles, while all N-terminal extensions of CA abolished cylinder formation. Spherical particles with heterogeneous diameters or amorphous protein aggregates were observed instead. Extending CA by 5 amino acids was sufficient to convert the assembly phenotype to spherical particles. Sequences C-terminal of CA were not required for sphere formation. Proteolytic cleavage of N-terminally extended CA proteins prior to in vitro assembly led to the formation of cylindrical particles, while proteolysis of in vitro assembly products caused disruption of spheres but not formation of cylinders. In vitro assembly of CA and extended CA proteins in the presence of cyclophilin A (CypA) at a CA-to-CypA molar ratio of 10:1 yielded significantly longer cylinders and heterogeneous spheres, while higher concentrations of CypA completely disrupted particle formation. We conclude that the spherical shape of immature HIV particles is determined by the presence of an N-terminal extension on the CA domain and that core condensation during virion maturation requires the liberation of the N terminus of CA.     

Critical role of conserved hydrophobic residues within the major homology region in mature retroviral capsid assembly

During retroviral maturation, the CA protein undergoes dramatic structural changes and establishes unique intermolecular interfaces in the mature capsid shell that are different from those that existed in the immature precursor. The most conserved region of CA, the major homology region (MHR), has been implicated in both immature and mature assembly, although the precise contribution of the MHR residues to each event has been largely undefined. To test the roles of specific MHR residues in mature capsid assembly, an in vitro system was developed that allowed for the first-time formation of Rous sarcoma virus CA into structures resembling authentic capsids. The ability of CA to assemble organized structures was destroyed by substitutions of two conserved hydrophobic MHR residues and restored by second-site suppressors, demonstrating that these MHR residues are required for the proper assembly of mature capsids in addition to any role that these amino acids may play in immature particle assembly. The defect caused by the MHR mutations was identified as an early step in the capsid assembly process. The results provide strong evidence for a model in which the hydrophobic residues of the MHR control a conformational reorganization of CA that is needed to initiate capsid assembly and suggest that the formation of an interdomain interaction occurs early during maturation.     

HIV-1壳体蛋白的结构及其病毒样颗粒疫苗

人类免疫缺陷病毒(HIV)的壳体蛋白(CA)在HIV病毒的组装和成熟过程中起着至关重要的作用。近年来,壳体蛋白的体外表达及其疫苗的研制成了HIV各项研究的焦点。由于壳体蛋白具有较好的的保守性,用其制得的疫苗也会提供比包膜蛋白更为广泛的免疫保护力。另外若将CA在体外表达成一个颗粒状结构,会增强其免疫原性,可以使疫苗发挥出更大的效力。     

Hepatitis E virus capsid protein assembles in 4M urea in the presence of salts

The hepatitis E virus (HEV) capsid protein has been demonstrated to be able to assemble into particles in vitro. However, this process and the mechanism of protein–protein interactions during particle assembly remain unclear. In this study, we investigated the assembly mechanism of HEV structural protein subunits, the capsid protein p239 (aa368–606), using analytical ultracentrifugation. It was the first to observe that the p239 can form particles in 4M urea as a result of supplementation with salt, including ammonium sulfate [(NH₄)₂SO₄], sodium sulfate (Na₂SO₄), sodium chloride (NaCl), and ammonium chloride (NH₄Cl). Interestingly, it is the ionic strength that determines the efficiency of promoting particle assembly. The assembly rate was affected by temperature and salt concentration. When (NH₄)₂SO₄ was used, assembling intermediates of p239 with sedimentation coefficient values of approximately 5 S, which were mostly dodecamers, were identified for the first time. A highly conserved 28‐aa region (aa368–395) of p239 was found to be critical for particle assembly, and the hydrophobic residues Leu³⁷², Leu³⁷⁵, and Leu³⁹⁵of p239 was found to be critical for particle assembly, which was revealed by site‐directed mutagenesis. This study provides new insights into the assembly mechanism of native HEV, and contributes a valuable basis for further investigations of protein assembly by hydrophobic interactions under denaturing conditions.     

The structural biology of HIV assembly

HIV assembly and replication proceed through the formation of morphologically distinct immature and mature viral capsids that are organized by the Gag polyprotein (immature) and by the fully processed CA protein (mature). The Gag polyprotein is composed of three folded polypeptides (MA, CA, and NC) and three smaller peptides (SP1, SP2, and p6) that function together to coordinate membrane binding and Gag-Gag lattice interactions in immature virions. Following budding, HIV maturation is initiated by proteolytic processing of Gag, which induces conformational changes in the CA domain and results in the assembly of the distinctive conical capsid. Retroviral capsids are organized following the principles of fullerene cones, and the hexagonal CA lattice is stabilized by three distinct interfaces. Recently identified inhibitors of viral maturation act by disrupting the final stage of Gag processing, or by inhibiting the formation of a critical intermolecular CA-CA interface in the mature capsid. Following release into a new host cell, the capsid disassembles and host cell factors can potently restrict this stage of retroviral replication. Here, we review the structures of immature and mature HIV virions, focusing on recent studies that have defined the global organization of the immature Gag lattice, identified sites likely to undergo conformational changes during maturation, revealed the molecular structure of the mature capsid lattice, demonstrated that capsid architectures are conserved, identified the first capsid assembly inhibitors, and begun to uncover the remarkable biology of the mature capsid.     

Polymorphism in the assembly of polyomavirus capsid protein VP1.

Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid.     

Rationally designed interfacial peptides are efficient in vitro inhibitors of HIV-1 capsid assembly with antiviral activity

Virus capsid assembly constitutes an attractive target for the development of antiviral therapies; a few experimental inhibitors of this process for HIV-1 and other viruses have been identified by screening compounds or by selection from chemical libraries. As a different, novel approach we have undertaken the rational design of peptides that could act as competitive assembly inhibitors by mimicking capsid structural elements involved in intersubunit interfaces. Several discrete interfaces involved in formation of the mature HIV-1 capsid through polymerization of the capsid protein CA were targeted. We had previously designed a peptide, CAC1, that represents CA helix 9 (a major part of the dimerization interface) and binds the CA C-terminal domain in solution. Here we have mapped the binding site of CAC1, and shown that it substantially overlaps with the CA dimerization interface. We have also rationally modified CAC1 to increase its solubility and CA-binding affinity, and designed four additional peptides that represent CA helical segments involved in other CA interfaces. We found that peptides CAC1, its derivative CAC1M, and H8 (representing CA helix 8) were able to efficiently inhibit the in vitro assembly of the mature HIV-1 capsid. Cocktails of several peptides, including CAC1 or CAC1M plus H8 or CAI (a previously discovered inhibitor of CA polymerization), or CAC1M+H8+CAI, also abolished capsid assembly, even when every peptide was used at lower, sub-inhibitory doses. To provide a preliminary proof that these designed capsid assembly inhibitors could eventually serve as lead compounds for development of anti-HIV-1 agents, they were transported into cultured cells using a cell-penetrating peptide, and tested for antiviral activity. Peptide cocktails that drastically inhibited capsid assembly in vitro were also able to efficiently inhibit HIV-1 infection ex vivo. This study validates a novel, entirely rational approach for the design of capsid assembly interfacial inhibitors that show antiviral activity.     

Assembly of human immunodeficiency virus (HIV) antigens on bacteriophage T4: a novel in vitro approach to construct multicomponent HIV vaccines

Bacteriophage T4 capsid is an elongated icosahedron decorated with 155 copies of Hoc, a nonessential highly antigenic outer capsid protein. One Hoc monomer is present in the center of each major capsid protein (gp23*) hexon. We describe an in vitro assembly system which allows display of HIV antigens, p24-gag, Nef, and an engineered gp41 C-peptide trimer, on phage T4 capsid surface through Hoc-capsid interactions. In-frame fusions were constructed by splicing the human immunodeficiency virus (HIV) genes to the 5' or 3' end of the Hoc gene. The Hoc fusion proteins were expressed, purified, and displayed on hoc(-) phage particles in a defined in vitro system. Single or multiple antigens were efficiently displayed, leading to saturation of all available capsid binding sites. The displayed p24 was highly immunogenic in mice in the absence of any external adjuvant, eliciting strong p24-specific antibodies, as well as Th1 and Th2 cellular responses with a bias toward the Th2 response. The phage T4 system offers new direction and insights for HIV vaccine development with the potential to increase the breadth of both cellular and humoral immune responses.     

A small molecule inhibits and misdirects assembly of hepatitis B virus capsids

Hepatitis B virus (HBV) capsids play an important role in viral nucleic acid metabolism and other elements of the virus life cycle. Misdirection of capsid assembly (leading to formation of aberrant particles) may be a powerful approach to interfere with virus production. HBV capsids can be assembled in vitro from the dimeric capsid protein. We show that a small molecule, bis-ANS, binds to capsid protein, inhibiting assembly of normal capsids and promoting assembly of noncapsid polymers. Using equilibrium dialysis to investigate binding of bis-ANS to free capsid protein, we found that only one bis-ANS molecule binds per capsid protein dimer, with an association energy of -28.0 +/- 2.0 kJ/mol (-6.7 +/- 0.5 kcal/mol). Bis-ANS inhibited in vitro capsid assembly induced by ionic strength as observed by light scattering and size exclusion chromatography. The binding energy of bis-ANS for capsid protein calculated from assembly inhibition data was -24.5 +/- 0.9 kJ/mol (-5.9 +/- 0.2 kcal/mol), essentially the same binding energy observed in studies of unassembled protein. These data indicate that capsid protein bound to bis-ANS did not participate in assembly; this mechanism of assembly inhibition is analogous to competitive or noncompetitive inhibition of enzymes. While assembly of normal capsids is inhibited, our data suggest that bis-ANS leads to formation of noncapsid polymers. Evidence of aberrant polymers was identified by light scattering and electron microscopy. We propose that bis-ANS acts as a molecular "wedge" that interferes with normal capsid protein geometry and capsid formation; such wedges may represent a new class of antiviral agent.     

Rubella virus capsid associates with host cell protein p32 and localizes to mitochondria

Togavirus nucleocapsids have a characteristic icosahedral structure and are composed of multiple copies of a capsid protein complexed with genomic RNA. The assembly of rubella virus nucleocapsids is unique among togaviruses in that the process occurs late in virus assembly and in association with intracellular membranes. The goal of this study was to identify host cell proteins which may be involved in regulating rubella virus nucleocapsid assembly through their interactions with the capsid protein. Capsid was used as bait to screen a CV1 cDNA library using the yeast two-hybrid system. One protein that interacted strongly with capsid was p32, a cellular protein which is known to interact with other viral proteins. The interaction between capsid and p32 was confirmed using a number of different in vitro and in vivo methods, and the site of interaction between these two proteins was shown to be at the mitochondria. Interestingly, overexpression of the rubella virus structural proteins resulted in clustering of the mitochondria in the perinuclear region. The p32-binding site in capsid is a potentially phosphorylated region that overlaps the viral RNA-binding domain of capsid. Our results are consistent with the possibility that the interaction of p32 with capsid plays a role in the regulation of nucleocapsid assembly and/or virus-host interactions.     

Retrovirus capsid protein assembly arrangements

During retrovirus particle assembly and morphogenesis, the retrovirus structural (Gag) proteins organize into two different arrangements: an immature form assembled by precursor Gag (PrGag) proteins; and a mature form, composed of proteins processed from PrGag. Central to both Gag protein arrangements is the capsid (CA) protein, a domain of PrGag, which is cleaved from the precursor to yield a mature Gag protein composed of an N-terminal domain (NTD), a flexible linker region, and a C-terminal domain (CTD). Because Gag interactions have proven difficult to examine in virions, a number of investigations have focused on the analysis of structures assembled in vitro. We have used electron microscope (EM) image reconstruction techniques to examine assembly products formed by two different CA variants of both human immunodeficiency virus type 1 (HIV-1) and the Moloney murine leukemia virus (M-MuLV). Interestingly, two types of hexameric protein arrangements were observed for each virus type. One organizational scheme featured hexamers composed of putative NTD dimer subunits, with sharing of subunits between neighbor hexamers. The second arrangement used apparent NTD monomers to coordinate hexamers, involved no subunit sharing, and employed putative CTD interactions to connect hexamers. Conversion between the two assembly forms may be achieved by making or breaking the proposed symmetric NTD dimer contacts in a process that appears to mimic viral morphogenesis.     

Production and purification of SV40 major capsid protein (VP1) in Escherichia coli strains deficient for the GroELS chaperone machine

Production of the major capsid protein of SV40, VP1, is of great interest for the study on capsid assembly in vitro. Production of soluble His6-VP1 in Escherichia coli strains deficient in the GroELS chaperone machine was substantially higher than in the wild-type strain. The His6-VP1 produced in a groEL mutant strain was readily purified. The protein was able to form higher-order structures as evidenced by analysis of the soluble fraction by gel filtration, by sedimentation in sucrose gradient, and by electron microscopy. We propose the use of groE mutants for the production of the major capsid protein of SV40 and perhaps also other papovaviruses.     

Retroviral Capsid Assembly: A Role for the CA Dimer in Initiation

In maturing retroviral virions, CA protein assembles to form a capsid shell that is essential for infectivity. The structure of the two folded domains [N-terminal domain (NTD) and C-terminal domain (CTD)] of CA is highly conserved among various retroviruses, and the capsid assembly pathway, although poorly understood, is thought to be conserved as well. In vitro assembly reactions with purified CA proteins of the Rous sarcoma virus (RSV) were used to define factors that influence the kinetics of capsid assembly and provide insights into underlying mechanisms. CA multimerization was triggered by multivalent anions providing evidence that in vitro assembly is an electrostatically controlled process. In the case of RSV, in vitro assembly was a well-behaved nucleation-driven process that led to the formation of structures with morphologies similar to those found in virions. Isolated RSV dimers, when mixed with monomeric protein, acted as efficient seeds for assembly, eliminating the lag phase characteristic of a monomer-only reaction. This demonstrates for the first time the purification of an intermediate on the assembly pathway. Differences in the intrinsic tryptophan fluorescence of monomeric protein and the assembly-competent dimer fraction suggest the involvement of the NTD in the formation of the functional dimer. Furthermore, in vitro analysis of well-characterized CTD mutants provides evidence for assembly dependence on the second domain and suggests that the establishment of an NTD-CTD interface is a critical step in capsid assembly initiation. Overall, the data provide clear support for a model whereby capsid assembly within the maturing virion is dependent on the formation of a specific nucleating complex that involves a CA dimer and is directed by additional virion constituents.     

Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro

The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.     

Human immunodeficiency virus type 1 capsid formation in reticulocyte lysates.

The Gag polyprotein of human immunodeficiency virus (HIV) (Pr55Gag) contains sufficient information to direct particle assembly events when expressed within tissue culture cells. HIV Gag proteins normally form particles at a plasma membrane assembly site, in a manner analogous to that of the type C avian and mammalian leukemia/sarcoma viruses. It has not previously been demonstrated that immature HIV capsids can form without budding through an intact cellular membrane. In this study, a rabbit reticulocyte lysate translation reaction was used to recreate HIV capsid formation in vitro. Production of HIV-1 Pr55Gag and of a matrix-deleted Gag construct resulted in the formation of a subset of Gag protein structures with an equilibrium density of 1.15 g/ml. Gel filtration chromatography revealed these Gag protein structures to be larger than 2 x 10(6) Da, consistent with the formation of large multimers or capsids. These Gag protein structures were protease sensitive in the absence of detergent, indicating that they did not contain a complete lipid envelope. Spherical structures were detected by electron microscopy within the reticulocyte lysate reaction mixtures and appeared essentially identical to immature HIV capsids or retrovirus-like particles. These results demonstrate that the HIV Gag protein is capable of producing immature capsids in a cell-free reaction and that such capsids lack a complete lipid envelope.