Modeling and Molecular Dynamics of the 3D Structure of the HPV16 E7 Protein and Its Variants.

The oncogenic potential of high-risk human papillomavirus (HPV) is predicated on the production of the E6 and E7 oncoproteins, which are responsible for disrupting the control of the cell cycle. Epidemiological studies have proposed that the presence of the N29S and H51N variants of the HPV16 E7 protein is significantly associated with cervical cancer. It has been suggested that changes in the amino acid sequence of E7 variants may affect the oncoprotein 3D structure; however, this remains uncertain. An analysis of the structural differences of the HPV16 E7 protein and its variants (N29S and H51N) was performed through homology modeling and structural refinement by molecular dynamics simulation. We propose, for the first time, a 3D structure of the E7 reference protein and two of Its variants (N29S and H51N), and conclude that the mutations induced by the variants in N29S and H51N have a significant influence on the 3D structure of the E7 protein of HPV16, which could be related to the oncogenic capacity of this protein.

Human Papillomavirus G-Rich Regions as Potential Antiviral Drug Targets.

Herein, we report, for the first time, the screening of several ligands in terms of their ability to bind and stabilize G-quadruplexes (G4) found in seven human Papillomavirus (HPV) genomes. Using a variety of biophysical assays, HPV G-quadruplexes were shown to possess a high degree of structural polymorphism upon ligand binding, which may have an impact on transcription, replication, and viral protein production. A sequence found in high-risk HPV16 genotype folds into multiple non-canonical DNA structures; it was converted into a major G4 conformation upon interaction with a well-characterized highly selective G4 ligand, PhenDC3, which may have an impact on the viral infection. Likewise, HPV57 and 58, which fold into multiple G4 structures, were found to form single stable complexes in the presence of two other G4 ligands, C8 and pyridostatin, respectively. In addition, one of the selected compounds, the acridine derivative C8, demonstrated a significant antiviral effect in HPV18-infected organotypic raft cultures. Altogether, these results indicate that targeting HPV G4s may be an alternative route for the development of novel antiviral therapies.

Human Papillomavirus 16 Capsids Mediate Nuclear Entry during Infection.

Infectious human papillomavirus 16 (HPV16) L1/L2 pseudovirions were found to remain largely intact during vesicular transport to the nucleus. By electron microscopy, capsids with a diameter of 50 nm were clearly visible within small vesicles attached to mitotic chromosomes and to a lesser extent within interphase nuclei, implying nuclear disassembly. By confocal analysis, it was determined that nuclear entry of assembled L1 is dependent upon the presence of the minor capsid protein, L2, but independent of encapsidated DNA. We also demonstrate that L1 nuclear localization and mitotic chromosome association can occur in vivo in the murine cervicovaginal challenge model of HPV16 infection. These findings challenge the prevailing concepts of PV uncoating and disassembly. More generally, they document that a largely intact viral capsid can enter the nucleus within a transport vesicle, establishing a novel mechanism by which a virus accesses the nuclear cellular machinery.IMPORTANCE Papillomaviruses (PVs) comprise a large family of nonenveloped DNA viruses that include HPV16, among other oncogenic types, the causative agents of cervical cancer. Delivery of the viral DNA into the host cell nucleus is necessary for establishment of infection. This was thought to occur via a subviral complex following uncoating of the larger viral capsid. In this study, we demonstrate that little disassembly of the PV capsid occurs prior to nuclear delivery. These surprising data reveal a previously unrecognized viral strategy to access the nuclear replication machinery. Understanding viral entry mechanisms not only increases our appreciation of basic cell biological pathways but also may lead to more effective antiviral interventions.

[Preparation and Cryo-EM Structure Determination of Human Papillomavirus 16 Pseudovirion Derived from Suspension-adapted HEK293 Cells].

The goals of this study were to establish a scalable production method to prepare human papillomavirus(HPV)16pseudovirus (PsV) using suspension-adapted HEK-293 FT cells and to improve the purification efficiency of HPV PsV. Furthermore, we aimed to solve the cryo-electron microscopy (cryo-EM) structure of HPV16 PsV. The suspension f HEK-293 FT cells were generated from adherent cells by a stepwise decrease in serum content and the addition of an anti-clumping agent during culturing. The resultant HEK-293 FT suspension cells were transfected with an L1/L2 expression vector and pN31-EGFP plasmid to generate HPV16 PsV in the Wave Bioreactor. Following cell lysis,HPV16 PsV was purified by sucrose density gradient and subsequent CsCl iso-density gradient ultra-centrifugation The final titer of HPV16 PsV was 8.2 × 10(5) TCID(50)/µL. Purified HPV16 PsV was comfirmed to as contain L1 and L2protein by western blotting, and the L1 concentration was determined to be 156.0 µg/mL by quantitative ELISA. Finally, a FEI Tecnai G2F30 electron microscope and AUTO3 DEM were used to solve the cryoEM structure of HPV16 PsV at a resolution of 14 Å.The structure shows that HPV16 PsV exists as a T=7dicosahedral lattice, with a diameter of 600 Å. These results will be beneficial for neutralization assays and for anti-sera for HPV vaccines, the high-resolution structure determination of HPV16 PsV, and the investigation of interactions between HPV L1 and L2.

Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization.

Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays.

Maturation of the human papillomavirus 16 capsid.

Papillomaviruses are a family of nonenveloped DNA viruses that infect the skin or mucosa of their vertebrate hosts. The viral life cycle is closely tied to the differentiation of infected keratinocytes. Papillomavirus virions are released into the environment through a process known as desquamation, in which keratinocytes lose structural integrity prior to being shed from the surface of the skin. During this process, virions are exposed to an increasingly oxidative environment, leading to their stabilization through the formation of disulfide cross-links between neighboring molecules of the major capsid protein, L1. We used time-lapse cryo-electron microscopy and image analysis to study the maturation of HPV16 capsids assembled in mammalian cells and exposed to an oxidizing environment after cell lysis. Initially, the virion is a loosely connected procapsid that, under in vitro conditions, condenses over several hours into the more familiar 60-nm-diameter papillomavirus capsid. In this process, the procapsid shrinks by ~5% in diameter, its pentameric capsomers change in structure (most markedly in the axial region), and the interaction surfaces between adjacent capsomers are consolidated. A C175S mutant that cannot achieve normal inter-L1 disulfide cross-links shows maturation-related shrinkage but does not achieve the fully condensed 60-nm form. Pseudoatomic modeling based on a 9-Å resolution reconstruction of fully mature capsids revealed C-terminal disulfide-stabilized "suspended bridges" that form intercapsomeric cross-links. The data suggest a model in which procapsids exist in a range of dynamic intermediates that can be locked into increasingly mature configurations by disulfide cross-linking, possibly through a Brownian ratchet mechanism. Importance: Human papillomaviruses (HPVs) cause nearly all cases of cervical cancer, a major fraction of cancers of the penis, vagina/vulva, anus, and tonsils, and genital and nongenital warts. HPV types associated with a high risk of cancer, such as HPV16, are generally transmitted via sexual contact. The nonenveloped virion of HPVs shows a high degree of stability, allowing the virus to persist in an infectious form in environmental fomites. In this study, we used cryo-electron microscopy to elucidate the structure of the HPV16 capsid at different stages of maturation. The fully mature capsid adopts a rigid, highly regular structure stabilized by intermolecular disulfide bonds. The availability of a pseudoatomic model of the fully mature HPV16 virion should help guide understanding of antibody responses elicited by HPV capsid-based vaccines.

[An ultrastructural study of the cervix epitelium infected with the human papillomavirus types 16 and 18 before and after treatment with contrasting thermo-laser therapy].

The results of the ultrastructural study of the epithelium of the patient cervix infected by the human papillomavirus (HPV) types 16 and 18 before and after treatment by contrasting thermo-laser therapy (CTLT) are presented. It was shown in this work that 1.5 and 6 months after treatment HPV DNA was not detected in the biopsy and the smear of the cervix using the polymerase chain reaction (PCR). In the ultrathin sections, the structure of the epithelial cells from the biopsy after treatment corresponded to norm. There was effective elimination of HPV types 16 and 18 as Induces by CTLT method.

Annexin A2 and S100A10 regulate human papillomavirus type 16 entry and intracellular trafficking in human keratinocytes.

Human papillomaviruses (HPVs) cause benign and malignant tumors of the mucosal and cutaneous epithelium. The initial events regulating HPV infection impact the establishment of viral persistence, which is requisite for malignant progression of HPV-infected lesions. However, the precise mechanisms involved in HPV entry into host cells, including the cellular factors regulating virus uptake, are not clearly defined. We show that HPV16 exposure to human keratinocytes initiates epidermal growth factor receptor (EGFR)-dependent Src protein kinase activation that results in phosphorylation and extracellular translocation of annexin A2 (AnxA2). HPV16 particles interact with AnxA2 in association with S100A10 as a heterotetramer at the cell surface in a Ca(2+)-dependent manner, and the interaction appears to involve heparan-sulfonated proteoglycans. We show multiple lines of evidence that this interaction promotes virus uptake into host cells. An antibody to AnxA2 prevents HPV16 internalization, whereas an antibody to S100A10 blocks infection at a late endosomal/lysosomal site. These results suggest that AnxA2 and S100A10 have separate roles during HPV16 binding, entry, and trafficking. Our data additionally imply that AnxA2 and S100A10 may be involved in regulating the intracellular trafficking of virus particles prior to nuclear delivery of the viral genome.

Human papillomavirus and cervical cancer.

Cervical cancer is caused by human papillomavirus infection. Most human papillomavirus infection is harmless and clears spontaneously but persistent infection with high-risk human papillomavirus (especially type 16) can cause cancer of the cervix, vulva, vagina, anus, penis, and oropharynx. The virus exclusively infects epithelium and produces new viral particles only in fully mature epithelial cells. Human papillomavirus disrupts normal cell-cycle control, promoting uncontrolled cell division and the accumulation of genetic damage. Two effective prophylactic vaccines composed of human papillomavirus type 16 and 18, and human papillomavirus type 16, 18, 6, and 11 virus-like particles have been introduced in many developed countries as a primary prevention strategy. Human papillomavirus testing is clinically valuable for secondary prevention in triaging low-grade cytology and as a test of cure after treatment. More sensitive than cytology, primary screening by human papillomavirus testing could enable screening intervals to be extended. If these prevention strategies can be implemented in developing countries, many thousands of lives could be saved.

Disassembly and reassembly improves morphology and thermal stability of human papillomavirus type 16 virus-like particles.

Recombinant human papillomavirus (HPV) 16 L1 protein self-assembles into virus-like particles (VLPs) with diameters of 40 to 60 nm, which are key components in prophylactic HPV vaccines. Marked improvement in morphology and thermal stability on VLP disassembly and reassembly was demonstrated at production scale. Differential scanning calorimetry showed enhanced conformational stability as indicated by the unfolding temperatures and peak heights/areas. Cloud point studies indicated (1) a much lower propensity for post-reassembly VLPs to aggregate during a time course study and (2) much higher cloud point temperatures. In-solution atomic force microscopy showed more uniform size distribution and fully closed particles, with evidence of virion-like assembly revealed by the structural details from a single particle image. Similar approaches for the reassembly of other recombinant VLPs with intrinsic conformational switches would be expected to improve the particle properties and render nanoparticles more suitable for use as vaccines or therapeutics. FROM THE CLINICAL EDITOR: The authors of this study demonstrated that recombinant human papillomavirus 16 L1 protein self-assembles into virus-like particles (VLPs) with marked improvement in morphology and thermal stability on VLP disassembly and reassembly at production scale. This is expected to render these nanoparticles more suitable for use as vaccines or therapeutics.

Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity.

BACKGROUND: Human papillomavirus (HPV) vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R), has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs). RESULTS: VLPs were subjected to post-purification disassembly and reassembly (D/R) treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs) H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5) was greatly reduced. Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres. CONCLUSIONS: D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical properties of VLPs and to correlate the observed changes at the molecular level. Mapping of known antibody epitopes to the homology model explains the changes in antibody reactivity upon D/R. In particular, the H16.H5 epitope is partially occluded by intercapsomeric interactions involving the L1 C-terminal arm. The homology model allows a more precise mapping of antibody epitopes. This work provides a better understanding of VLPs in current vaccines and could guide the design of improved vaccines or therapeutics.

Simple and convenient chromatography-based methods for purifying the pseudovirus of human papillomavirus type 58.

The currently available purification protocol for human papillomavirus (HPV) pseudovirus (PsV), as a substitute for the native HPV virion, utilizes Optiprep gradients (OG), which require costly equipment such as ultracentrifuges, and 4-7 h of working time, and cannot cope with large PsV samples. To develop a convenient method for purifying HPV type 58 PsV (HPV58 PsV) we have examined the use of heparin chromatography (HC) and cation-exchange chromatography (CC), which utilize open column systems and do not require expensive equipment. We confirmed that the PsVs resulting from HC and CC have correctly assembled conformations and are neutralized by anti-HPV58 PsV mouse serum, indicating that their antigenic characteristics make them suitable to substitute for native HPV58 virion. The recoveries of infectious PsV resulting from HC and CC were 39% and 11%, respectively, while that from OG was 58%. The two new purification methods are advantageous with respect to working time (only 30 min) and expense over the OG method, and have considerable potential for large scale purification.

Production of immunogenic human papillomavirus-16 major capsid protein derived virus like particles.

BACKGROUND & OBJECTIVE: Recombinant DNA technology allows expression of the human papillomavirus (HPV) major capsid protein (L1) in heterologous expression systems and the recombinant protein self assembles to virus-like particles (VLP). We took up this study to produce recombinant HPV-16 L1 in yeast, establish the process of recombinant L1 derived VLP preparation and develop an ELISA using VLP as the antigen for serological evaluation of anti HPV-16 L1 antibody status. METHODS: Complete HPV-16 L1 was amplified from genomic DNA of an esophageal cancer biopsy, cloned and the protein was expressed in a galactose-inducible Saccharomyces cerevisiae expression system. Self assembled VLP was purified by a two-step density gradient centrifugation process and the VLP preparation used to test its suitability in developing an ELISA. RESULTS: The recombinant protein was predominantly a ~55 KD species with distinct immunoreactivity and formed VLP as confirmed by electron microscopy. An ELISA using the VLP showed its efficacy in appropriate immunoreactivity to serum/plasma IgG. INTERPRETATION & CONCLUSION: Recombinant HPV-16 capsid protein derived VLP was produced and the VLP antigen based ELISA can be used to probe serological association of HPV with different clinical conditions. The VLP technology can be improved further and harnessed for future vaccine development efforts in the country.

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris.

Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin-sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines.

[Production of human papillomavirus type 16 virus-like particles and its immunogenicity].

HPV16 L1 gene was amplified from HPV16 positive vaginal secretion sample by PCR, and inserted into pTO-T7 to obtain the recombinant expression vector pTO-T7-HPV16-L1. Then, the pTO-T7-HPV16-L1 was transformed into E. coil strain ER2566 and the recombinant protein HPV16 L1 was expressed in soluble form. After purification by ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic interaction chromatography, the recombinant protein HPV16 L1 had a purity of more than 98%. By removing DTT, purified HPV16 L1 proteins self-assembled in vitro into VLPs with the diameter of 50 nm. The vaccination experiments on experimental animals showed the VLPs could elicit high titer of neutralizing antibodies against HPV 16. HPV16 VLPs with high immunogenicity and high purity can be produced easily and effectively from an E. coli expression system in the study, and thus can be used in structure investigation and HPV16 vaccine development.

Human papillomavirus type 16 exists in bacteria isolated from cervical cancer biopsies.

This study investigated the association between infectious microbes and persistent infection with human papillomavirus type 16 (HPV-16) in cervical cancer. Bacterial strains (identified as Enterococcus, Staphylococcus, Bacillus and Corynebacterium, based on their partial 16S rDNA sequence) were HPV-16 positive from 12 out of 14 cervical cancer biopsies. Total DNA was isolated from the four bacterial strains, and HPV-16 genes and genome were detected using polymerase chain reaction (PCR) and Southern blotting. RNA transcripts for HPV-16 E6 and L1 genes were detected in total bacterial RNA samples using reverse transcription-PCR, and HPV-16 L1 protein expression was detected in bacterial cells by Western blotting and immunocolloidal gold electron microscopy. The presence of virus particles in bacterial cells was demonstrated by transmission electron microscopy. The results suggest that bacteria carrying HPV-16 could provide a potential explanation for how infectious microbes contribute to the progression from HPV-16 infection to cervical cancer.

Insertion of a targeting peptide on capsid surface loops of human papillomavirus type-16 virus-like particles mediate elimination of anti-dsDNA Abs-producing B cells with high efficiency.

The purpose of this study was to design chimeric human papillomavirus type-16 L1 virus-like particles (VLPs) and to explore the potential capacity of elimination to anti-dsDNA antibody-producing B cells. To test it, VLPs were achieved by combination of human papillomavirus type-16 L1 proteins inserted into a targeting peptide (DWEYSVWLSN) and plasmids encoding diphtheria toxin A ligand. Additionally, VLPs were cocultured with target cells to assess the killing efficiency by lactate dehydrogenase assay in vitro. Lastly, lupus-prone (BWF1) mice vaccinated with VLPs were used as a model to assess the killing efficiency in vivo. The results showed that the VLPs were constructed successfully, and possessed the potential of killing anti-dsDNA antibody-producing B cells with high efficiency. The findings indicate the possibility that the VLPs ablate autoreactive B cells represents a novel strategy in the immunotherapy of autoantibody-mediated diseases.

Clathrin- and caveolin-independent entry of human papillomavirus type 16--involvement of tetraspanin-enriched microdomains (TEMs).

BACKGROUND: Infectious entry of human papillomaviruses into their host cells is an important step in the viral life cycle. For cell binding these viruses use proteoglycans as initial attachment sites. Subsequent transfer to a secondary receptor molecule seems to be involved in virus uptake. Depending on the papillomavirus subtype, it has been reported that entry occurs by clathrin- or caveolin-mediated mechanisms. Regarding human papillomavirus type 16 (HPV16), the primary etiologic agent for development of cervical cancer, clathrin-mediated endocytosis was described as infectious entry pathway. METHODOLOGY/PRINCIPAL FINDINGS: Using immunofluorescence and infection studies we show in contrast to published data that infectious entry of HPV16 occurs in a clathrin- and caveolin-independent manner. Inhibition of clathrin- and caveolin/raft-dependent endocytic pathways by dominant-negative mutants and siRNA-mediated knockdown, as well as inhibition of dynamin function, did not impair infection. Rather, we provide evidence for involvement of tetraspanin-enriched microdomains (TEMs) in HPV16 endocytosis. Following cell attachment, HPV16 particles colocalized with the tetraspanins CD63 and CD151 on the cell surface. Notably, tetraspanin-specific antibodies and siRNA inhibited HPV16 cell entry and infection, confirming the importance of TEMs for infectious endocytosis of HPV16. CONCLUSIONS/SIGNIFICANCE: Tetraspanins fulfill various roles in the life cycle of a number of important viral pathogens, including human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, their involvement in endocytosis of viral particles has not been proven. Our data indicate TEMs as a novel clathrin- and caveolin-independent invasion route for viral pathogens and especially HPV16.

Intrinsic fluorescence as an analytical probe of virus-like particle assembly and maturation.

The assembly and maturation of the human papillomavirus (HPV) virus-like particle (VLP) has been monitored by measuring the intrinsic fluorescence intensity using excitation at 290nm and emission at 350nm. The assay was validated to eliminate error due to photo-bleaching, adsorption, and precipitation. Intrinsic fluorescence intensity dropped during both assembly and maturation phases. The decrease during assembly had a second-order dependence on capsomere concentration, as previously observed using light scattering. During post-assembly structural modification the decrease had a first-order dependence on capsomere concentration. Intrinsic fluorescence spectroscopy complements light scattering methodologies for monitoring assembly and enables kinetics of maturation to be observed. The role of environmental factors such as the presence of oxidized glutathione in facilitation of faster and more complete maturation was monitored in real time. Intrinsic fluorescence is a rugged methodology that could be applied to monitoring VLP assembly and maturation unit operations during HPV vaccine manufacturing.

A quantitative description of in vitro assembly of human papillomavirus 16 virus-like particles.

The full-length human papillomavirus 16 major capsid protein L1 is expressed in Saccharomyces cerevisiae as virus-like particles (VLPs). However, yeast-expressed human papillomavirus 16 particles are irregular in shape and are prone to aggregate. When disassembled and reassembled, the resulting particles have improved stability and solubility. We have examined VLP dissociation and reassembly to define the important features of the assembly mechanism. We found that the VLPs rapidly disassemble at pH 8.2 and low ionic strength in the presence of low concentrations of reducing agents. The pH dependence of assembly kinetics and extent of assembly under reducing conditions were differentially sensitive to ionic strength. Assembly at pH 5.2 was very fast and led to heavily aggregated particles. This sort of kinetic trap is expected for overinitiated assembly. We observed that reassembly at pH 6.2, 7.2, and 8.2 yielded regular particles over a broad range of ionic strength. At these three pH values, assembly was quantitative at 1 M NaCl. At pH 7.2, much more than at pH 6.2 or pH 8.2, assembly decreased monotonically with ionic strength. The free energy of association ranged from -8 to -10 kcal/mol per pentamer. The effect of pH on assembly was further investigated by examining dissociation of reassembled particles. Though indistinguishable by negative stain electron microscopy, particles assembled at pH 7.2 disassembled slower than pH 5.2, 6.2, or 8.2 VLPs. We hypothesize that pH 7.2 assembly reactions lead to formation of particles with conformationally different interactions.