RNA interference suppression of A100A4 reduces the growth and metastatic phenotype of human renal cancer cells via NF-kB-dependent MMP-2 and bcl-2 pathway.

BACKGROUND AND AIM: S100A4 is a well established marker and mediator of metastatic disease, but the exact mechanisms responsible for the metastasis promoting effects are less well defined. We tested a hypothesis that the S100A4 gene plays a role in the proliferation and invasiveness of human renal cancer cells (RCC) and may be associated with its metastatic spread. MATERIALS AND METHODS: The small interference RNA vector pcDNA3.1-S100A4 siRNA was transfected in to the human renal cancer cell lines ACHN, Ketr-3, OS-RC-2, CaKi-2 and HTB-47, then treated with ABT-737 or BB94. Cell apoptosis and cell viability was detected by flow cytometry and MTT assay. Matrigel was used for cell motility and invasion assay. MMP-2, bcl-2 and S100A4 was detected by RT-PCR and western blot assay. NF-kB subunit p65 activity was detected by confocal microscopy assay. We then determine the effect S100A4 sliencing on tumor growth, lung metastasis development in vivo. Immunohistochemistry was used to detected the expression of S100A4, bcl-2, MMP-2, p65 and CD31. RESULTS: S100A4 silencing in ACHN cells by RNA interference significantly inhibited NF-kB and NF-kB-mediated MMP-2 and bcl-2 activation and cellular migration, proliferation, and promoted apoptosis. Furthermore, re-expression of S100A4 in S100A4-siRNA-transfected ACHN cells by transient S100A4 cDNA transfection restored the NF-kB and NF-kB-mediated MMP-2 and bcl-2 activation and their high migratory and cellular proliferative ability. An inhibitor ABT-737 (the Bcl-2 antagonist targets Bcl-2) against Bcl-2 suppressed cellular proliferation and promoted apoptosis induced by S100A4 re-expression in S100A4-siRNA-transfected ACHN cells. A inhibitor BB94 against MMPs to neutralize MMP-2 protein suppressed cellular invasion and migration induced by S100A4 re-expression in S100A4-siRNA-transfected ACHN cells. In the prevention model, S100A4 silencing inhibited primary tumor growth by (tumor weight) (76 ± 8%) and (tumor volum) (78 ± 4%) respectively and promoted apoptosis and the formation of lung metastases was inhibited by 89% (p < 0.01). Microvascular density was reduced by 70% (p < 0.01). In addition, S100A4 sliencing inhibited the expression of S100A4 in vivo, followed by the NF-kB, MMP-2 and bcl-2 suppression. CONCLUSIONS: We conclude that S100A4 plays a crucial role in proliferation and migratory/invasive processes in human RCC by a mechanism involving activation of NF-kB-bcl-2 and NF-kB-MMP-2 pathway.

INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.

AIMS/HYPOTHESIS: We aimed to generate human embryonic stem cell (hESC) reporter lines that would facilitate the characterisation of insulin-producing (INS⁺) cells derived in vitro. METHODS: Homologous recombination was used to insert sequences encoding green fluorescent protein (GFP) into the INS locus, to create reporter cell lines enabling the prospective isolation of viable INS⁺ cells. RESULTS: Differentiation of INS(GFP/w) hESCs using published protocols demonstrated that all GFP⁺ cells co-produced insulin, confirming the fidelity of the reporter gene. INS-GFP⁺ cells often co-produced glucagon and somatostatin, confirming conclusions from previous studies that early hESC-derived insulin-producing cells were polyhormonal. INS(GFP/w) hESCs were used to develop a 96-well format spin embryoid body (EB) differentiation protocol that used the recombinant protein-based, fully defined medium, APEL. Like INS-GFP⁺ cells generated with other methods, those derived using the spin EB protocol expressed a suite of pancreatic-related transcription factor genes including ISL1, PAX6 and NKX2.2. However, in contrast with previous methods, the spin EB protocol yielded INS-GFP⁺ cells that also co-expressed the beta cell transcription factor gene, NKX6.1, and comprised a substantial proportion of monohormonal INS⁺ cells. CONCLUSIONS/INTERPRETATION: INS(GFP/w) hESCs are a valuable tool for investigating the nature of early INS⁺ progenitors in beta cell ontogeny and will facilitate the development of novel protocols for generating INS⁺ cells from differentiating hESCs.

High resolution genomic analysis of sporadic breast cancer using array-based comparative genomic hybridization.

INTRODUCTION: Genomic aberrations in the form of subchromosomal DNA copy number changes are a hallmark of epithelial cancers, including breast cancer. The goal of the present study was to analyze such aberrations in breast cancer at high resolution. METHODS: We employed high-resolution array comparative genomic hybridization with 4,134 bacterial artificial chromosomes that cover the genome at 0.9 megabase resolution to analyze 47 primary breast tumors and 18 breast cancer cell lines. RESULTS: Common amplicons included 8q24.3 (amplified in 79% of tumors, with 5/47 exhibiting high level amplification), 1q32.1 and 16p13.3 (amplified in 66% and 57% of tumors, respectively). Moreover, we found several positive correlations between specific amplicons from different chromosomes, suggesting the existence of cooperating genetic loci. Queried by gene, the most frequently amplified kinase was PTK2 (79% of tumors), whereas the most frequently lost kinase was PTK2B (hemizygous loss in 34% of tumors). Amplification of ERBB2 as measured by comparative genomic hybridization (CGH) correlated closely with ERBB2 DNA and RNA levels measured by quantitative PCR as well as with ERBB2 protein levels. The overall frequency of recurrent losses was lower, with no region lost in more than 50% of tumors; the most frequently lost tumor suppressor gene was RB1 (hemizygous loss in 26% of tumors). Finally, we find that specific copy number changes in cell lines closely mimicked those in primary tumors, with an overall Pearson correlation coefficient of 0.843 for gains and 0.734 for losses. CONCLUSION: High resolution CGH analysis of breast cancer reveals several regions where DNA copy number is commonly gained or lost, that non-random correlations between specific amplicons exist, and that specific genetic alterations are maintained in breast cancer cell lines despite repeat passage in tissue culture. These observations suggest that genes within these regions are critical to the malignant phenotype and may thus serve as future therapeutic targets.

Constitutive and regulated expression of processed insulin following in vivo hepatic gene transfer.

To test whether hepatocytes engineered in vivo can serve as surrogate beta cells by similarly secreting mature insulin in a glucose-sensitive manner, we prepared adenoviral vectors encoding wild-type proinsulin (hIns-wt), a modified proinsulin cleavable by the ubiquitously expressed protease furin (hIns-M3), or each of the two beta cell specific pro-insulin convertases PC2 and PC3. Following a detailed in vitro characterization of the proteins produced by our vectors, we infected the liver and, for comparison, the muscle of a chemically induced murine model of type I diabetes. Insulin expression from the transduced tissues was extensively characterized and showed to be constitutive rather than regulated. To obtain regulated expression, we placed expression of hIns-M3 under the control of the dimerizer-inducible transcription system. Hormone secretion from mouse liver was negligible in the absence of the dimerizer drug rapamycin, was inducible in a dose-dependent manner upon its administration, and reversible following drug withdrawal. These data confirm liver as a promising target for in vivo expression of processed insulin. While suggesting that hepatocytes cannot provide authentic glucose-responsive regulation, these results demonstrate that pharmacological regulation is a promising alternative route to the controlled delivery of insulin following hepatic gene transfer.

Adenoviral vector transfection into the pulmonary epithelium after cecal ligation and puncture in rats.

BACKGROUND: Adenoviral-targeted gene delivery to respiratory epithelium can augment production of specific proteins. Therefore, it may be valuable in treating the acute respiratory distress syndrome. The authors tested the hypothesis that adenoviral vector uptake after cecal ligation and double puncture in rats, an animal model of the acute respiratory distress syndrome, is higher than that observed in controls that did not undergo operation ("nonoperated") or those that underwent a sham operation ("sham-operated"). METHODS: Adenoviruses expressing green fluorescent protein or Lac-Z were delivered into the lungs of anesthetized rats via tracheal catheter. Animals were killed 24 or 48 h later. Histopathology and green fluorescent protein expression were examined using light of fluorescence microscopy. Cellular localization of Lac-Z was determined with electron microscopy or semithin sectioning. Viral receptor density and localization were determined using imunoblotting and immunohistochemistry. RESULTS: After cecal ligation and double puncture, rats were hypoxic and tachypneic. Alveoli were segmentally consolidated, contained proteinaceous debris and neutrophils, and had thickened septa. Administration of adenoviruses to rats that were sham-operated or underwent cecal ligation and double puncture resulted in high levels of marker protein expression in cells lining alveoli. Use of 3 x 10(11) plaque-forming units instead of 3 x 10(12) plaque-forming units resulted in similar levels of green fluorescent protein expression with negligible viral-mediated lymphocytic infiltration. Semithin section and electron microscopy revealed expression primarily localized to type II alveolar cells. Abundance of alpha(v)beta3 integrins and human coxsackie-adenovirus receptor (receptors that modulate viral attachment and internalization) was increased after cecal ligation and double puncture, predominantly in type II pneumocytes. CONCLUSIONS: Cecal ligation and double puncture induces histologic and functional changes consistent with the acute respiratory distress syndrome, increases surface expression of viral receptors, and enhances adenoviral-mediated gene transfer.

Adenoviral vector-mediated insulin gene transfer in the mouse pancreas corrects streptozotocin-induced hyperglycemia.

Therapy for type 1 diabetes consists of tight blood glucose (BG) control to minimize complications. Current treatment relies on multiple insulin injections or an insulin pump placement, beta-cell or whole pancreas transplantation. All approaches have significant limitations and have led to the realization that novel treatment strategies are needed. Pancreatic acinar cells have features that make them a good target for insulin gene transfer. They are not subject to autoimmune attack, a problem with pancreas or islets transplantation, they are avidly transduced by recombinant adenoviral vectors, and capable of exporting a variety of peptides into the portal circulation. Recombinant adenoviral vectors were engineered to express either wild-type or furin-modified human insulin cDNA (AdCMVhInsM). Immunodeficient mice were made diabetic with streptozotocin and injected intrapancreatically with the vectors. BG and blood insulin levels have normalized after administration of AdCMVhInsM. Immunohistochemistry and electron microscopy showed the presence of insulin in acinar cells throughout the pancreas and localization of insulin molecules to acinar cell vesicles. The data clearly establish a relationship between intrapancreatic vector administration, decreased BG and elevated blood insulin levels. The findings support the use of pancreatic acinar cells to express and secrete insulin into the blood stream.

Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors.

The innate immune response to intraportally infused adenoviral vector was evaluated in rhesus monkeys. A first-generation adenovirus-expressing lacZ (Ad-lacZ) was administered at a dose just below that which causes severe morbidity. The response to vector was evaluated for the initial 24 h following infusion. Clinical findings during this time were primarily limited to petechiae, consistent with the development of thrombocytopenia and biochemical evidence of disseminated intravascular coagulation. Serum transaminases were elevated and a lymphopenia developed. Tracking of fluorescent-labeled vector demonstrated distribution to macrophages and dendritic cells of the spleen and Kupffer cells of the liver. A systemic release of the cytokine IL-6 occurred soon after vector infusion. Analysis of splenic cells revealed acute activation of macrophages and dendritic cells followed by massive apoptosis. Bone marrow cultures demonstrated normal erythroid and primitive progenitors with a significant decrease in myeloid progenitors. Similar findings, except the abnormality in bone marrow cultures, were observed in monkeys who received an identical dose of Ad-lacZ in which vector genes were inactivated with psoralen and UV irradiation. These data suggest that inadvertent targeting of antigen-presenting cells following intraportal infusion of vector leads to a systemic cytokine syndrome which may be triggered by the viral capsid proteins.

Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo.

Traditional gene therapy vectors have demonstrated limited utility for treatment of chronic lung diseases such as cystic fibrosis (CF). Herein we describe a vector based on a Filovirus envelope protein-pseudotyped HIV vector, which we chose after systematically evaluating multiple strategies. The vector efficiently transduces intact airway epithelium from the apical surface, as demonstrated in both in vitro and in vivo model systems. This shows the potential of pseudotyping in expanding the utility of lentiviral vectors. Pseudotyped lentiviral vectors may hold promise for the treatment of CF.

Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo.

Recombinant adeno-associated virus (AAV) holds much promise for human gene therapy. While evidence indicates that AAV mediates long-term gene transfer in several different tissues, difficulty in preparing and purifying this viral vector in large quantities remains a major obstacle for evaluating AAV vectors in clinical trials. The current method of purification, based on sedimentation through cesium chloride, is not scaleable and yields product of insufficient quality. In this article we report a new technique for purifying AAV, using a fully closed two-column chromatography system. Yields of AAV vectors purified by this method are high, potency is increased, and the purity of column-purified preparations is substantially improved. We previously reported a novel method to generate AAV based on an AAV Rep/Cap-containing cell line (B50) and an Ad-AAV hybrid virus, which is amenable to scale-up in bioreactors. By combining the new, fully scaleable purification process we report here with the B50/hybrid production method, it would be feasible to prepare AAV vectors to the scale and purity required for clinical and potential commercial applications.

Development of a rapid method for the PEGylation of adenoviruses with enhanced transduction and improved stability under harsh storage conditions.

PEGylation is the covalent attachment of activated monomethoxy poly(ethylene) glycols (MPEGs) to free lysine groups of therapeutic proteins. This technology has enhanced the physical stability of proteins and ablated humoral immune responses generated against them. In this study, adenoviral vectors were modified with MPEGs activated by cyanuric chloride, succinimidyl succinate, and tresyl chloride. Under proper buffering conditions, reactions were complete within 2 hr. Transduction efficiency of PEGylated adenoviruses was not compromised by neutralizing antibodies to native adenovirus in vitro. These preparations retained titers that were significantly greater than those of the unconjugated virus after storage at 42, 25, 4, and -20 degrees C. Stability profiles of PEGylated preparations at -20 degrees C suggest that glycerol could be eliminated from formulations without significant loss of viral titer. PEGylated adenoviruses produced a two- to threefold increase in transduction in the lung when administered by intratracheal injection and a fivefold increase in transduction in the liver when administered intravenously.

Functional differences between keratins of stratified and simple epithelia.

Dividing populations of stratified and simple epithelial tissues express keratins 5 and 14, and keratins 8 and 18, respectively. It has been suggested that these keratins form a mechanical framework important to cellular integrity, since their absence gives rise to a blistering skin disorder in neonatal epidermis, and hemorrhaging within the embryonic liver. An unresolved fundamental issue is whether different keratins perform unique functions in epithelia. We now address this question using transgenic technology to express a K16-14 hybrid epidermal keratin transgene and a K18 simple epithelial keratin transgene in the epidermis of mice null for K14. Under conditions where the hybrid epidermal keratin restored a wild-type phenotype to newborn epidermis, K18 partially but not fully rescued. The explanation does not appear to reside in an inability of K18 to form 10-nm filaments with K5, which it does in vitro and in vivo. Rather, it appears that the keratin network formed between K5 and K18 is deficient in withstanding mechanical stress, leading to perturbations in the keratin network in regions of the skin that are subjected either to natural or to mechanically induced trauma. Taken together, these findings suggest that the loss of a type I epidermal keratin cannot be fully compensated by its counterpart of simple epithelial cells, and that in vivo, all keratins are not equivalent.

Integrators of epidermal growth and differentiation: distinct functions for beta 1 and beta 4 integrins.

Mammalian epithelia are critically dependent on interactions with components in the underlying basal lamina for proper morphogenesis and function. Substratum attachment is essential for survival, proliferation, movement, and differentiation; detachment compromises the cell's ability to perform these functions, often resulting in human disease. Interactions with the extracellular matrix are mediated through transmembrane integrin receptors that transmit signals to the cytoskeleton and to signaling molecules within the proliferating cells of the epithelium. In the past year, novel insights have emerged regarding the specific role of integrins in their attachment to extracellular matrix and in their signal transduction pathways within the epidermis.

An unexpected localization of basonuclin in the centrosome, mitochondria, and acrosome of developing spermatids.

Basonuclin is a zinc finger protein that was thought to be restricted to keratinocytes of stratified squamous epithelia. In epidermis, basonuclin is associated with the nuclei of mitotically active basal cells but not in terminally differentiating keratinocytes. We report here the isolation of a novel form of basonuclin, which we show is also expressed in stratified epithelia. Most unexpectedly, we find both forms in testis, where a surprising localization pattern was uncovered. While basonuclin RNA expression occurs in mitotically active germ cells, protein was not detected until the meiotic stage, where basonuclin localized to the appendage of the distal centriole of spermatocytes and spermatids. Near the end of spermiogenesis, basonuclin also accumulated in the acrosome and mitochondrial sheath surrounding the flagellum. Intriguingly, a perfect six-amino acid residue mitochondrial targeting sequence (Komiya, T., N. Hachiya, M. Sakaguchi, T. Omura, and K. Mihara. 1994. J. Biol. Chem. 269:30893-30897; Shore, G.C., H.M. McBride, D.G. Millar, N.A. Steenaart, and M. Nguyen. 1995. Eur. J. Biochem. 227: 9-18; McBride, H.M., I.S. Goping, and G.C. Shore. 1996. J. Cell. Biol. 134:307-313) is present in basonuclin 1a but not in the 1b form. Moreover, three distinct affinity-purified peptide antibodies gave this unusual pattern of basonuclin antibody staining, which was confirmed by cell fractionation studies. Our findings suggest a unique role for basonuclin in centrosomes within the developing spermatid, and a role for one of the protein forms in germ cell mitochondrial function. Its localization with the acrosome suggests that it may also perform a special function during or shortly after fertilization.

Progressive kidney degeneration in mice lacking tensin.

Tensin is a focal adhesion phosphoprotein that binds to F-actin and contains a functional Src homology 2 domain. To explore the biological functions of tensin, we cloned the mouse tensin gene, determined its program of expression, and used gene targeting to generate mice lacking tensin. Even though tensin is expressed in many different tissues during embryogenesis, tensin null mice developed normally and appeared healthy postnatally for at least several months. Over time, -/- mice became frail because of abnormalities in their kidneys, an organ that expresses high levels of tensin. Mice with overt signs of weakness exhibited signs of renal failure and possessed multiple large cysts in the proximal kidney tubules, but even in tensin null mice with normal blood analysis, cysts were prevalent. Ultrastructurally, noncystic areas showed typical cell-matrix junctions that readily labeled with antibodies against other focal adhesion molecules. In abnormal regions, cell-matrix junctions were disrupted and tubule cells lacked polarity. Taken together, our data imply that, in the kidney, loss of tensin leads to a weakening, rather than a severing, of focal adhesion. All other tissues appeared normal, suggesting that, in most cases, tensin's diverse functions are redundant and may be compensated for by other focal adhesion proteins.

The genetic basis of epidermolysis bullosa simplex with mottled pigmentation.

Epidermolysis bullosa simplex (EBS) is a group of autosomal dominant skin diseases characterized by blistering, due to mechanical stress-induced degeneration of basal epidermal cells. It is now well-established that the three major subtypes of EBS are genetic disorders of the basal epidermal keratins, keratin 5 (K5) and keratin 14 (K14). Here we show that a rare subtype, referred to as EBS with mottled pigmentation (MP), is also a disorder of these keratins. Affected members of two seemingly unrelated families with EBS-MP had a C to T point mutation in the second base position of codon 24 of one of two K5 alleles, leading to a Pro: Leu mutation. This mutation was not present in unaffected members nor in 100 alleles from normal individuals. Linkage analyses mapped the defect to this type II keratin gene (peak logarithm of odds score at phi = 0 of 3.9), which is located on chromosome 12q11-q13. This provides strong evidence that this mutation is responsible for the EBS-MP phenotype. Only conserved between K5 and K6, and not among any of the other type II keratins, Pro-24 is in the nonhelical head domain of K5, and only mildly perturbs the length of 10-nm keratin filaments assembled in vitro. However, this part of the K5 head domain is likely to protrude on the filament surface, perhaps leading to additional aberrations in intermediate filament architecture and/or in melanosome distribution that are seen ultrastructurally in patients with the mutation.

An essential cytoskeletal linker protein connecting actin microfilaments to intermediate filaments.

Typified by rapid degeneration of sensory neurons, dystonia musculorum mice have a defective BPAG1 gene, known to be expressed in epidermis. We report a neuronal splice form, BPAG1n, which localizes to sensory axons. Both isoforms have a coiled-coil rod, followed by a carboxy domain that associates with intermediate filaments. However, the amino terminus of BPAG1n differs from BPAG1e in that it contains a functional actin-binding domain. In transfected cells, BPAG1n coaligns neurofilaments and microfilaments, establishing this as a cytoskeletal protein interconnecting actin and intermediate filament cytoskeletons. In BPAG1 null mice, axonal architecture is markedly perturbed, consistent with a failure to tether neurofilaments to the actin cytoskeleton and underscoring the physiological relevance of this protein.

Beta4 integrin is required for hemidesmosome formation, cell adhesion and cell survival.

The integrin heterodimer alpha 6 beta 4 is expressed in many epithelia and in Schwann cells. In stratified epithelia, alpha 6 beta 4 couple with BPAG1-e and BPAG2 to form hemidesmosomes, attaching externally to laminin and internally to the keratin cytoskeleton. To explore the function of this atypical integrin, and its relation to conventional actin-associated integrins, we targeted the removal of the beta 4 gene in mice. Tissues that express alpha 6 beta 4 are grossly affected. Stratified tissues are devoid of hemidesmosomes, display only a very fragile attachment to the basal lamina, and exhibit signs of degeneration and tissue disorganization. Simple epithelia which express alpha 6 beta 4 are also defective in adherence, even though they do not form hemidesmosomes. In the absence of beta 4, alpha 6 is dramatically downregulated, and other integrins do not appear to compensate for the loss of this heterodimer. These data have important implications for understanding integrin function in cell-substratum adhesion, cell survival and differentiation, and for understanding the role of alpha 6 beta 4 in junctional epidermolysis bullosa, an often lethal human disorder with pathology similar to our mice.

Mice expressing a mutant desmosomal cadherin exhibit abnormalities in desmosomes, proliferation, and epidermal differentiation.

Desmogleins are members of the cadherin superfamily which form the core of desmosomes. In vitro studies indicate that the cytoplasmic domain of desmogleins associates with plakoglobin; however, little is known about the role of this domain in desmosome recognition or assembly in vivo, or about the possible relation of desmoglein mutations to epidermal differentiation and disease. To address these questions we used transgenic mouse technology to produce an NH2-terminally truncated desmoglein (Pemphigus Vulgaris Antigen or Dsg3) in cells known to express its wild-type counterpart. Within 2 d, newborn transgenic animals displayed swelling of their paws, flakiness on their back, and blackening of the tail tip. When analyzed histologically and ultrastructurally, widening of intercellular spaces and disruption of desmosomes were especially striking in the paws and tail. Desmosomes were reduced dramatically in number and were smaller and often peculiar in structure. Immunofluorescence and immunoelectron microscopy revealed no major abnormalities in localization of hemidesmosomal components, but desmosomal components organized aberrantly, resulting in a loss of ultrastructure within the plaque. In regions where desmosome loss was prevalent but where some adhesive structures persisted, the epidermis was thickened, with a marked increase in spinous and stratum corneum layers, variability in granular layer thickness, and parakeratosis in some regions. Intriguingly, a dramatic increase in cell proliferation was also observed concomitant with biochemical changes, including alterations in integrin expression, known to be associated with hyperproliferation. An inflammatory response was also detected in some skin regions. Collectively, these findings demonstrate that a mutation in a desmoglein can perturb epidermal cell-cell adhesion, triggering a cascade of changes in the skin.

Role of the C terminus Gag protein in human immunodeficiency virus type 1 virion assembly and maturation.

Lentiviral Gag polyproteins have a proline-rich protein, p6, at their C terminus. There are conflicting reports about the function of p6 in virus release. In the present work, mutants that affect p6 of human immunodeficiency virus type 1 (HIV-1) Gag polyprotein were constructed and analysed. None of the mutants prevented virus release completely; however, detachment of budding particles was less efficient as evidenced by electron microscopy. Virions of the p6 truncation mutant B2TAA had a significantly reduced number of Pol proteins (p66, p51 and p34) and an increased amount of incompletely processed Gag proteins compared with the parental virus. A mutation that altered the cleavage site between p6 and p1 did not significantly affect virus assembly, virus release or protein processing with the exception of cleavage between p6 and p1. However, virions of this mutant (B2P6C) exhibited irregular-shaped core structures that were distinct from the cone-shaped core structure seen in the parental virion. B2P6C mutant virus was non-infectious in CD4+ T cells. These results suggest that mutations in p6 affect efficient detachment of budding particles from the cell surface. Proper cleavage between p6 and p1 may be critical for the formation of the distinctive cone-shaped core structure of HIV-1 virions.