A Bivalent, Spherical Virus-Like Particle Vaccine Enhances Breadth of Immune Responses against Pathogenic Ebola Viruses in Rhesus Macaques.

The 2013-2016 Ebola outbreak in West Africa led to accelerated efforts to develop vaccines against these highly virulent viruses. A live, recombinant vesicular stomatitis virus-based vaccine has been deployed in outbreak settings and appears highly effective. Vaccines based on replication-deficient adenovirus vectors either alone or in combination with a multivalent modified vaccinia Ankara (MVA) Ebola vaccine also appear promising and are progressing in clinical evaluation. However, the ability of current live vector-based approaches to protect against multiple pathogenic species of Ebola is not yet established, and eliciting durable responses may require additional booster vaccinations. Here, we report the development of a bivalent, spherical Ebola virus-like particle (VLP) vaccine that incorporates glycoproteins (GPs) from Zaire Ebola virus (EBOV) and Sudan Ebola virus (SUDV) and is designed to extend the breadth of immunity beyond EBOV. Immunization of rabbits with bivalent Ebola VLPs produced antibodies that neutralized all four pathogenic species of Ebola viruses and elicited antibody-dependent cell-mediated cytotoxicity (ADCC) responses against EBOV and SUDV. Vaccination of rhesus macaques with bivalent VLPs generated strong humoral immune responses, including high titers of binding, as well as neutralizing antibodies and ADCC responses. VLP vaccination led to a significant increase in the frequency of Ebola GP-specific CD4 and CD8 T cell responses. These results demonstrate that a novel bivalent Ebola VLP vaccine elicits strong humoral and cellular immune responses against pathogenic Ebola viruses and support further evaluation of this approach as a potential addition to Ebola vaccine development efforts.IMPORTANCE Ebola outbreaks result in significant morbidity and mortality in affected countries. Although several leading candidate Ebola vaccines have been developed and advanced in clinical testing, additional vaccine candidates may be needed to provide protection against different Ebola species and to extend the durability of protection. A novel approach demonstrated here is to express two genetically diverse glycoproteins on a spherical core, generating a vaccine that can broaden immune responses against known pathogenic Ebola viruses. This approach provides a new method to broaden and potentially extend protective immune responses against Ebola viruses.

HIV-1 Envelope Glycoprotein Trafficking through the Endosomal Recycling Compartment Is Required for Particle Incorporation.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) encodes specific trafficking signals within its long cytoplasmic tail (CT) that regulate incorporation into HIV-1 particles. Rab11-family interacting protein 1C (FIP1C) and Rab14 are host trafficking factors required for Env particle incorporation, suggesting that Env undergoes sorting from the endosomal recycling compartment (ERC) to the site of particle assembly on the plasma membrane. We disrupted outward sorting from the ERC by expressing a C-terminal fragment of FIP1C (FIP1C560-649) and examined the consequences on Env trafficking and incorporation into particles. FIP1C560-649 reduced cell surface levels of Env and prevented its incorporation into HIV-1 particles. Remarkably, Env was trapped in an exaggerated perinuclear ERC in a CT-dependent manner. Mutation of either the Yxxϕ endocytic motif or the YW795 motif in the CT prevented Env trapping in the ERC and restored incorporation into particles. In contrast, simian immunodeficiency virus SIVmac239 Env was not retained in the ERC, while substitution of the HIV-1 CT for the SIV CT resulted in SIV Env retention in this compartment. These results provide the first direct evidence that Env traffics through the ERC and support a model whereby HIV-1 Env is specifically targeted to the ERC prior to FIP1C- and CT-dependent outward sorting to the particle assembly site on the plasma membrane.IMPORTANCE The HIV envelope protein is an essential component of the viral particle. While many aspects of envelope protein structure and function have been established, the pathway it follows in the cell prior to reaching the site of particle assembly is not well understood. The envelope protein has a very long cytoplasmic tail that interacts with the host cell trafficking machinery. Here, we utilized a truncated form of the trafficking adaptor FIP1C protein to arrest the intracellular transport of the envelope protein, demonstrating that it becomes trapped inside the cell within the endosomal recycling compartment. Intracellular trapping resulted in a loss of envelope protein on released particles and a corresponding loss of infectivity. Mutations of specific trafficking motifs in the envelope protein tail prevented its trapping in the recycling compartment. These results establish that trafficking to the endosomal recycling compartment is an essential step in HIV envelope protein particle incorporation.

Siglec-1 initiates formation of the virus-containing compartment and enhances macrophage-to-T cell transmission of HIV-1.

HIV-1 particles assemble and bud from the plasma membrane of infected T lymphocytes. Infected macrophages, in contrast, accumulate particles within an apparent intracellular compartment known as the virus-containing compartment or VCC. Many aspects of the formation and function of the VCC remain unclear. Here we demonstrate that VCC formation does not actually require infection of the macrophage, but can be reproduced through the exogenous addition of non-infectious virus-like particles or infectious virions to macrophage cultures. Particles were captured by Siglec-1, a prominent cell surface lectin that attaches to gangliosides on the lipid envelope of the virus. VCCs formed within infected macrophages were readily targeted by the addition of ganglioside-containing virus-like particles to the extracellular media. Depletion of Siglec-1 from the macrophage or depletion of gangliosides from viral particles prevented particle uptake into the VCC and resulted in substantial reductions of VCC volume. Furthermore, Siglec-1-mediated virion capture and subsequent VCC formation was required for efficient trans-infection of autologous T cells. Our results help to define the nature of this intracellular compartment, arguing that it is a compartment formed by particle uptake from the periphery, and that this compartment can readily transmit virus to target T lymphocytes. Inhibiting or eliminating the VCC may be an important component of strategies to reduce HIV transmission and to eradicate HIV reservoirs.

ROCK1 and LIM kinase modulate retrovirus particle release and cell-cell transmission events.

UNLABELLED: The assembly and release of retroviruses from the host cells require dynamic interactions between viral structural proteins and a variety of cellular factors. It has been long speculated that the actin cytoskeleton is involved in retrovirus production, and actin and actin-related proteins are enriched in HIV-1 virions. However, the specific role of actin in retrovirus assembly and release remains unknown. Here we identified LIM kinase 1 (LIMK1) as a cellular factor regulating HIV-1 and Mason-Pfizer monkey virus (M-PMV) particle release. Depletion of LIMK1 reduced not only particle output but also virus cell-cell transmission and was rescued by LIMK1 replenishment. Depletion of the upstream LIMK1 regulator ROCK1 inhibited particle release, as did a competitive peptide inhibitor of LIMK1 activity that prevented cofilin phosphorylation. Disruption of either ROCK1 or LIMK1 led to enhanced particle accumulation on the plasma membrane as revealed by total internal reflection fluorescence microscopy (TIRFM). Electron microscopy demonstrated a block to particle release, with clusters of fully mature particles on the surface of the cells. Our studies support a model in which ROCK1- and LIMK1-regulated phosphorylation of cofilin and subsequent local disruption of dynamic actin turnover play a role in retrovirus release from host cells and in cell-cell transmission events. IMPORTANCE: Viruses often interact with the cellular cytoskeletal machinery in order to deliver their components to the site of assembly and budding. This study indicates that a key regulator of actin dynamics at the plasma membrane, LIM kinase, is important for the release of viral particles for HIV as well as for particle release by a distantly related retrovirus, Mason-Pfizer monkey virus. Moreover, disruption of LIM kinase greatly diminished the spread of HIV from cell to cell. These findings suggest that LIM kinase and its dynamic modulation of the actin cytoskeleton in the cell may be an important host factor for the production, release, and transmission of retroviruses.

Rab11-FIP1C and Rab14 direct plasma membrane sorting and particle incorporation of the HIV-1 envelope glycoprotein complex.

The incorporation of the envelope glycoprotein complex (Env) onto the developing particle is a crucial step in the HIV-1 lifecycle. The long cytoplasmic tail (CT) of Env is required for the incorporation of Env onto HIV particles in T cells and macrophages. Here we identify the Rab11a-FIP1C/RCP protein as an essential cofactor for HIV-1 Env incorporation onto particles in relevant human cells. Depletion of FIP1C reduced Env incorporation in a cytoplasmic tail-dependent manner, and was rescued by replenishment of FIP1C. FIP1C was redistributed out of the endosomal recycling complex to the plasma membrane by wild type Env protein but not by CT-truncated Env. Rab14 was required for HIV-1 Env incorporation, and FIP1C mutants incapable of binding Rab14 failed to rescue Env incorporation. Expression of FIP1C and Rab14 led to an enhancement of Env incorporation, indicating that these trafficking factors are normally limiting for CT-dependent Env incorporation onto particles. These findings support a model for HIV-1 Env incorporation in which specific targeting to the particle assembly microdomain on the plasma membrane is mediated by FIP1C and Rab14.

Tetherin/BST-2 is essential for the formation of the intracellular virus-containing compartment in HIV-infected macrophages.

HIV-1 assembly and release occur at the plasma membrane in T lymphocytes, while intracellular sites of virus assembly or accumulation are apparent in macrophages. The host protein tetherin (BST-2) inhibits HIV release from the plasma membrane by retaining viral particles at the cell surface, but the role of tetherin at intracellular HIV assembly sites is unclear. We determined that tetherin is significantly upregulated upon macrophage infection and localizes to an intracellular virus-containing compartment (VCC). Tetherin localized at the virus-VCC membrane interface, suggesting that tetherin physically tethers virions in VCCs. Tetherin knockdown diminished and redistributed VCCs within macrophages and promoted HIV release and cell-cell transmission. The HIV Vpu protein, which downregulates tetherin from the plasma membrane, did not fully overcome tetherin-mediated restriction of particle release in macrophages. Thus, tetherin is essential for VCC formation and may account for morphologic differences in the apparent HIV assembly sites in macrophages versus T cells.

The intracellular virus-containing compartments in primary human macrophages are largely inaccessible to antibodies and small molecules.

HIV-1 assembly and release occurs at the plasma membrane of human T lymphocytes and model epithelial cell lines, whereas in macrophages intracellular sites of virus assembly or accumulation predominate. The origin of the intracellular virus-containing compartment (VCC) has been controversial. This compartment is enriched in markers of the multivesicular body, and has been described as a modified endosomal compartment. Several studies of this compartment have revealed the presence of small channels connecting to the plasma membrane, suggesting that instead of an endosomal origin the compartment is a modified plasma membrane compartment. If the compartment is accessible to the external environment, this would have important implications for antiviral immune responses and antiviral therapy. We performed a series of experiments designed to determine if the VCC in macrophages was open to the external environment and accessible to antibodies and small molecules. The majority of VCCs were found to be inaccessible to exogenously-applied antibodies to tetraspanins in the absence of membrane permeabilization, while tetraspanin staining was readily observed following membrane permeabilization. Cationized ferritin was utilized to stain the plasma membrane, and revealed that the majority of virus-containing compartments were inaccessible to ferritin. Low molecular weight dextrans could access only a very small percentage of VCCs, and these tended to be more peripheral compartments. We conclude that the VCCs in monocyte-derived human macrophages are heterogeneous, but the majority of VCCs are closed to the external environment.

The cytotoxic protein can induce autophagocytosis in addition to apoptosis in MCF-7 human breast cancer cells.

Phagocytic clearance of dying cells is found in many phagocytes. It has been shown that dying cells can be phagocytosed by other phagocytic cells through autophagic or apoptotic cellular death. To date, whether cancer cells have such phagocytic activity has not been studied. In this study, our data shows that RC-RNase can trigger cell death in human breast cancer MCF-7 cells through the apoptotic pathway. Interestingly, when treated with cytotoxic protein, the remaining MCF-7 cells can phagocytose the dying MCF-7 cells via autophagocytic activity, demonstrated directly by real-time image observation and electron microscopy analysis. To sum up, this study demonstrates for the first time that RC-RNase can trigger apoptosis and autophagocytosis in MCF-7 cancer cells.

Rana catesbeiana ribonuclease inhibits Japanese encephalitis virus (JEV) replication and enhances apoptosis of JEV-infected BHK-21 cells.

Rana catesbeiana ribonuclease (RC-RNase) is a cytotoxic and antitumor RNase isolated from the oocyte yolk granules of the bullfrog R. catesbeiana. Our previous studies have shown that RC-RNase possesses antitumor activity by activating proapoptotic caspases. Here, we demonstrate that RC-RNase also possesses antiviral activity. By using cell viability and caspase activation assays, we show that RC-RNase largely enhances apoptosis of Japanese encephalitis virus (JEV)-infected BHK-21 cells by activating caspase-3, caspase-8, and caspase-9. In addition, immunoblotting experiments revealed that JEV infection enhances the internalization of RC-RNase by cells. In sum, these results indicate that RC-RNase provides a beneficial effect on JEV-infected cells by enhancing apoptosis.

RC-RNase-induced cell death in estrogen receptor positive breast tumors through down-regulation of Bcl-2 and estrogen receptor.

RC-RNase exerts anti-cancer effects on many tumors. However, the mechanisms by which RC-RNase induces cytotoxicity in different tumor cells are unclear. Currently, estrogen receptor (ER)-positive and negative breast tumors are treated with RC-RNase. Our data demonstrate that RC-RNase induces cell death on ER-positive but not on ER-negative breast tumors. This study also shows that down-regulation of ER and Bcl-2 is found on RC-RNase-treated ER-positive breast tumors. Additionally, Bcl-2 overxpression can prevent ER-positive breast tumors from cell death treated with RC-RNase. In summary, this study demonstrates that RC-RNase-induced cell death of ER-positive breast tumors is through regulation of ER and Bcl-2.

Pseudovirion particles bearing native HIV envelope trimers facilitate a novel method for generating human neutralizing monoclonal antibodies against HIV.

Monomeric HIV envelope vaccines fail to elicit broadly neutralizing antibodies or to protect against infection. Neutralizing antibodies against HIV bind to native functionally active Env trimers on the virion surface. Gag-Env pseudovirions recapitulate the native trimer and could serve as an effective epitope presentation platform for study of the neutralizing antibody response in HIV-infected individuals. To address if pseudovirions can recapitulate native HIV virion epitope structures, we carefully characterized these particles, concentrating on the antigenic structure of the coreceptor binding site. By blue native gel shift assays, Gag-Env pseudovirions were shown to contain native trimers that were competent for binding to neutralizing monoclonal antibodies. In enzyme-linked immunosorbent assay, pseudovirions exhibited increased binding of known CD4-induced antibodies after addition of CD4. Using flow cytometric analysis, fluorescently labeled pseudovirions specifically identified a subset of antigen-specific B cells in HIV-infected subjects. Interestingly, the sequence of one of these novel human antibodies, identified during cloning of single HIV-specific B cells and designated 2C6, exhibited homology to mAb 47e, a known anti-CD4-induced coreceptor binding site antibody. The secreted monoclonal antibody 2C6 did not bind monomeric gp120, but specifically bound envelope on pseudovirions. A recombinant form of the antibody 2C6 acted as a CD4-induced epitope-specific antibody in neutralization assays, yet did not bind monomeric gp120. These findings imply specificity against a quaternary epitope presented on the pseudovirion envelope spike. These data demonstrate that Gag-Env pseudovirions recapitulate CD4 and coreceptor binding pocket antigenic structures and can facilitate identification of B-cell clones that secrete neutralizing antibodies.

Human immunodeficiency virus type-1 gag and host vesicular trafficking pathways.

The Gag protein of HIV-1 directs the particle assembly process. Gag recruits components of the cellular vesicular trafficking machinery in order to traverse the cytoplasm of the cell and reach the particle assembly site. The plasma membrane is the primary site of particle assembly in most cell types, while in macrophages an unusual intracellular membrane-bound compartment bearing markers of late endosomes and the plasma membrane is the predominant assembly site. Plasma membrane specificity of assembly may be directed by components of lipid rafts and the cytoplasmic leaflet component PI(4,5)P(2). Recent work has highlighted the role of adaptor protein complexes, protein sorting and recycling pathways, components of the multivesicular body, and cellular motor proteins in facilitating HIV assembly and budding. This review presents an overview of the relevant vesicular trafficking pathways and describes the individual components implicated in interactions with Gag.

Characterization of a myristoylated, monomeric HIV Gag protein.

The process of HIV assembly requires extensive homomultimerization of the Gag polyprotein on cellular membranes to generate the nascent particle bud. Here we generated a full-length, monomeric Gag polyprotein bearing mutations that eliminated multimerization in living cells as indicated by fluorescence resonance energy transfer (FRET). Monomeric Gag resembled non-myristoylated Gag in its weak membrane binding characteristics and lack of association with detergent-resistant membranes (DRMs or lipid rafts). Monomeric Gag failed to assemble virus-like particles, but was inefficiently rescued into particles by wildtype Gag through the influence of the matrix domain. The subcellular distribution of monomeric Gag was remarkably different than either non-myristoylated Gag or wildtype Gag. Monomeric Gag was found on intracellular membranes and at the plasma membrane, where it highlighted plasma membrane extensions and ruffles. This study indicates that monomeric Gag can traffic to assembly sites in the cell, where it interacts weakly with membranes.

Incorporation of human immunodeficiency virus type 1 reverse transcriptase into virus-like particles.

We demonstrate that a genetically engineered human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) composed mainly of p66 or p51 subunits can be incorporated into virus-like particles (VLPs) when coexpressed with HIV-1 Pr55(gag). VLP-associated RT exhibited a detergent-resistant association with immature cores during sucrose gradient equilibrium centrifugation, suggesting that RT is incorporated into VLPs. However, RT that retains downstream integrase (IN) is severely inhibited in terms of incorporation into VLPs. Results from immunofluorescence tests reveal that RT-IN is primarily localized at the perinuclear area and exhibits poor colocalization with Gag. IN removal leads to a redistribution of RT throughout the cytoplasm and improved RT incorporation into VLPs. Similar results were observed for RT-IN in which alanine was substituted for 186-Lys-Arg-Lys-188 residues of the IN putative nuclear localization signal, suggesting that IN karyophilic properties may partly account for the inhibitory effect of IN on RT incorporation. Although the membrane-binding capacity of RT was markedly reduced compared to that of wild-type Gag or Gag-Pol, the correlation of membrane-binding ability with particle incorporation efficiency was incomplete. Furthermore, we observed that membrane-binding-defective myristylation-minus RT can be packaged into VLPs at the same level as its normal myristylated counterpart. This suggests that the incorporation of RT into VLPs is independent of membrane affinity but very dependent on RT-Gag interaction. Results from a genetic analysis suggest that the Gag-interacting regions of RT mainly reside in the thumb subdomain and that the RT-binding domains of Gag are located in the matrix (MA) and p6 regions.

AP-3 directs the intracellular trafficking of HIV-1 Gag and plays a key role in particle assembly.

Gag proteins direct the process of retroviral particle assembly and form the major protein constituents of the viral core. The matrix region of the HIV-1 Gag polyprotein plays a critical role in the transport of Gag to the plasma membrane assembly site. Recent evidence indicates that Gag trafficking to late endosomal compartments, including multivesicular bodies, occurs prior to viral particle budding from the plasma membrane. Here we demonstrate that the matrix region of HIV-1 Gag interacts directly with the delta subunit of the AP-3 complex, and that this interaction plays an important functional role in particle assembly. Disruption of this interaction eliminated Gag trafficking to multivesicular bodies and diminished HIV particle formation. These studies illuminate an early step in retroviral particle assembly and provide evidence that the trafficking of Gag to late endosomes is part of a productive particle assembly pathway.

Synergism of Rana catesbeiana ribonuclease and IFN-gamma triggers distinct death machineries in different human cancer cells.

Rana catesbeiana ribonuclease (RC-RNase) possesses tumor-specific cytotoxicity, which can be synergized by IFN-gamma. However, it is unclear how RC-RNase and RC-RNase/IFN-gamma induce cell death. In this study, we use substrate cleavage assays to systematically investigate RC-RNase- and RC-RNase/IFN-gamma-induced caspase activation in HL-60, MCF-7, and SK-Hep-1 cells. We find that RC-RNase and RC-RNase/IFN-gamma induce mitochondria-mediated caspase activation in HL-60 and MCF-7 cells but not in SK-Hep-1 cells, although death of SK-Hep-1 cells is closely related to mitochondrial disruptions. Our findings provide evidence that RC-RNase and RC-RNase/IFN-gamma can kill different cancer cells by distinct mechanisms. Compared with onconase, RC-RNase seems to harbor a more specific anti-cancer activity.

High prevalence of phagocytic-resistant capsular serotypes of Klebsiella pneumoniae in liver abscess.

To better understand the role of capsular polysaccharide (CPS) K1 or K2 in Klebsiella pneumoniae liver abscess as well as the development of metastasis to eye, neutrophil phagocytosis of 70 CPS isolates including K1 (n = 23)/K2 (n = 10), non-K1/K2 (n = 37) was evaluated by flow cytometry, fluorescence imaging, and electron microscopy. K1/K2 isolates were significantly more resistant to phagocytosis (P < 0.0001) than non-K1/K2 isolates and displayed increased resistance to intracellular killing. Although mucoid phenotype (M-type) K1/K2 isolates were significantly more resistant to phagocytosis (P = 0.0029) than M-type non-K1/K2, no significant difference in the phagocytosis rate was observed between K1/K2 isolates with M-type and non-M-type (P = 0.0924). Mucoidy is an associated factor that was predominant in K1/K2 isolates, but which itself is not an independent influence on phagocytic resistance. The K1/K2 CPS proved significantly more resistant to phagocytosis than non-K1/K2 CPS in liver abscess isolates (P < 0.0001) and non-abscess isolates (P = 0.0001), suggesting that K1/K2 isolates were generally more virulent in both liver abscess and in non-liver abscess conditions. These findings indicate that resistance of CPS K1 or K2 K. pneumoniae to phagocytosis and intracellular killing presumably contributes to their high prevalence in liver abscess and uniquely in endophthalmitis.

Association of Japanese encephalitis virus NS3 protein with microtubules and tumour susceptibility gene 101 (TSG101) protein.

Previously reported findings by our group showed that non-structural protein 3 (NS3) of Japanese encephalitis virus (JEV) was localized mainly in the JEV-induced convoluted membrane (CM), which has been proposed to originate from rough endoplasmic reticulum (rER), Golgi apparatus or the trans-Golgi network (TGN), and serves as a reservoir for viral proteins during virus assembly. Earlier findings indicated that NS3 of Kunjin virus interacts with microtubules. In addition, one of the Golgi-associated proteins, tumour susceptibility protein 101 (TSG101), associates with microtubules and is required for budding of retroviral particles. To clarify the association of NS3 with microtubules or with TSG101 during JEV assembly, we applied immunofluorescence, co-immunoprecipitation and immunoelectron microscopic methods. Virus infection, as well as transfection with an NS2B-NS3 expression plasmid, induced microtubule rearrangement. When cells were treated with colchicine, which interferes with microtubule polymerization, NS3 still associated with tubulin and TSG101. Furthermore, tubulin and TSG101 were co-localized with NS3 in the CM by immunogold labelling. Our observations indicate that microtubules and TSG101 associate with NS3, which is incorporated into the JEV-induced structure during JEV replication.

Independent segregation of human immunodeficiency virus type 1 Gag protein complexes and lipid rafts.

Formation of human immunodeficiency virus type 1 (HIV-1) particles takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein. A functional assembly domain (the M domain) within the N-terminal portion of Pr55Gag mediates the interaction of Gag with cellular membranes. However, the determinants that provide specificity for assembly on the plasma membrane, as opposed to intracellular membranes, have not been identified. Recently, it was reported that Pr55Gag interacts with lipid raft microdomains of the plasma membrane. We sought to identify the domains within Pr55Gag that contribute to lipid raft association of Gag. Here we demonstrate that the I domain is required for interaction with detergent-resistant membrane fractions (DRMs). Mutation of key I-domain residues or loss of myristylation abrogated the association of Gag with DRMs. Thus, the I domain and the M domain combine to mediate Gag-lipid raft interactions as defined by these biochemical criteria. However, Gag protein complexes defined by flotation studies were much denser than classical lipid rafts, failed to incorporate classical lipid raft marker proteins, and were not disrupted by cholesterol extraction. Large sheets of Gag protein were identified in DRM fractions upon examination by electron microscopy. These results indicate that HIV-1 Pr55Gag forms detergent-resistant complexes at the cellular periphery that are distinct from lipid raft microdomains.

Induction of differentiation rescues HL-60 cells from Rana catesbeiana ribonuclease-induced cell death.

Rana catesbeiana ribonuclease (RC-RNase) exerted strong anti-tumor activity and its cytotoxicity was shown to correlate with differentiation stages of three different hepatoma cell lines. In this study, we demonstrate different RC-RNase cytotoxicity in undifferentiated HL-60 cells and in those that had been induced to differentiate by retinoic acid or dimethylsulfoxide. RC-RNase showed cytotoxicity in undifferentiated HL-60 cells, but not in HL-60 cells undergoing terminal differentiation. Furthermore, the caspase-9/caspase-3 pathway was activated when RC-RNase induced death in undifferentiated HL-60 cells and induction of differentiation led to a reversal of the caspase activation pathway.