Establishment of a novel human T-cell leukemia virus type 1 infection model using cell-free virus.

The primary mode of infection by human T-cell leukemia virus type 1 (HTLV-1) is cell-to-cell transmission during contact between infected cells and target cells. Cell-free HTLV-1 infections are known to be less efficient than infections with other retroviruses, and transmission of free HTLV-1 is considered not to occur in vivo. However, it has been demonstrated that cell-free HTLV-1 virions can infect primary lymphocytes and dendritic cells in vitro, and that virions embedded in biofilms on cell membranes can contribute to transmission. The establishment of an efficient cell-free HTLV-1 infection model would be a useful tool for analyzing the replication process of HTLV-1 and the clonal expansion of infected cells. We first succeeded in obtaining supernatants with high-titer cell-free HTLV-1 using a highly efficient virus-producing cell line. The HTLV-1 virions retained the structural characteristics of retroviruses. Using this cell-free infection model, we confirmed that a variety of cell lines and primary cultured cells can be infected with HTLV-1 and demonstrated that the provirus was randomly integrated into all chromosomes in the target cells. The provirus-integrated cell lines were HTLV-1-productive. Furthermore, we demonstrated for the first time that cell-free HTLV-1 is infectious in vivo using a humanized mouse model. These results indicate that this cell-free infection model recapitulates the HTLV-1 life cycle, including entry, reverse transcription, integration into the host genome, viral replication, and secondary infection. The new cell-free HTLV-1 infection model is promising as a practical resource for studying HTLV-1 infection.IMPORTANCECo-culture of infected and target cells is frequently used for studying HTLV-1 infection. Although this method efficiently infects HTLV-1, the cell mixture is complex, and it is extremely difficult to distinguish donor infected cells from target cells. In contrast, cell-free HTLV-1 infection models allow for more strict experimental conditions. In this study, we established a novel and efficient cell-free HTLV-1 infection model. Using this model, we successfully evaluated the infectivity titers of cell-free HTLV-1 as proviral loads (copies per 100 cells) in various cell lines, primary cultured cells, and a humanized mouse model. Interestingly, the HTLV-1-associated viral biofilms played an important role in enhancing the infectivity of the cell-free infection model. This cell-free HTLV-1 infection model reproduces the replication cycle of HTLV-1 and provides a simple, powerful, and alternative tool for researching HTLV-1 infection.

In vitro translation of virally-encoded replication polyproteins to recapitulate polyprotein maturation processes.

Single-stranded, positive-sense RNA viruses encode essential replication polyproteins which are composed of several domains. They are usually subjected to finely regulated proteolytic maturation processes to generate cleavage intermediates and end-products. Both polyproteins and maturation products play multiple key roles that ultimately allow synthesis of viral genome progeny. Despite the importance of these proteins in the course of viral replication, their structural properties, including the conformational changes regulating their numerous functions, are poorly described at the structural level. This lack of information is mainly due to the extreme difficulty to express large, membrane-bound, multi-domain proteins with criteria suitable for structural biology methods. To tackle this challenge, we have used a wheat-germ cell-free expression system. We firstly establish that this approach allows to synthesize viral polyproteins encoded by two unrelated positive-sense RNA viruses, a human norovirus and a plant tymovirus. Then, we demonstrate that these polyproteins are fully functional and are spontaneously auto-cleaved by their active protease domain, giving rise to natural maturation products. Moreover, we show that introduction of point mutations in polyproteins allows to inhibit the proteolytic maturation process of each virus. This allowed us to express and partially purify the uncleaved full-length norovirus polyprotein and the tymoviral RNA-dependent RNA polymerase. Thus, this study provides a powerful tool to obtain soluble viral polyproteins and their maturation products in order to conduct challenging structural biology projects and therefore solve unanswered questions.

Interviral Recombination between Plant, Insect, and Fungal RNA Viruses: Role of the Intracellular Ca2+/Mn2+ Pump.

Recombination is one of the driving forces of viral evolution. RNA recombination events among similar RNA viruses are frequent, although RNA recombination could also take place among unrelated viruses. In this paper, we have established efficient interviral recombination systems based on yeast and plants. We show that diverse RNA viruses, including the plant viruses tomato bushy stunt virus, carnation Italian ringspot virus, and turnip crinkle virus-associated RNA; the insect plus-strand RNA [(+)RNA] viruses Flock House virus and Nodamura virus; and the double-stranded L-A virus of yeast, are involved in interviral recombination events. Most interviral recombinants are minus-strand recombinant RNAs, and the junction sites are not randomly distributed, but there are certain hot spot regions. Formation of interviral recombinants in yeast and plants is accelerated by depletion of the cellular SERCA-like Pmr1 ATPase-driven Ca2+/Mn2+ pump, regulating intracellular Ca2+ and Mn2+ influx into the Golgi apparatus from the cytosol. The interviral recombinants are generated by a template-switching mechanism during RNA replication by the viral replicase. Replication studies revealed that a group of interviral recombinants is replication competent in cell-free extracts, in yeast, and in the plant Nicotiana benthamiana We propose that there are major differences among the viral replicases to generate and maintain interviral recombinants. Altogether, the obtained data promote the model that host factors greatly contribute to the formation of recombinants among related and unrelated viruses. This is the first time that a host factor's role in affecting interviral recombination is established.IMPORTANCE Viruses with RNA genomes are abundant, and their genomic sequences show astonishing variation. Genetic recombination in RNA viruses is a major force behind their rapid evolution, enhanced pathogenesis, and adaptation to their hosts. We utilized a previously identified intracellular Ca2+/Mn2+ pump-deficient yeast to search for interviral recombinants. Noninfectious viral replication systems were used to avoid generating unwanted infectious interviral recombinants. Altogether, interviral RNA recombinants were observed between plant and insect viruses, and between a fungal double-stranded RNA (dsRNA) virus and an insect virus, in the yeast host. In addition, interviral recombinants between two plant virus replicon RNAs were identified in N. benthamiana plants, in which the intracellular Ca2+/Mn2+ pump was depleted. These findings underline the crucial role of the host in promoting RNA recombination among unrelated viruses.

Murine FLT3 ligand-derived dendritic cell-mediated early immune responses are critical to controlling cell-free human T cell leukemia virus type 1 infection.

Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. We observed previously that depletion of dendritic cells (DCs) in CD11c-diphtheria toxin receptor transgenic mice followed by infection with cell-free virus led to greater proviral and Tax mRNA loads and diminished cellular immune response compared with mice infected with cell-associated virus. To understand the significance of these in vivo results and explore the host-pathogen interaction between DCs and cell-free HTLV-1, we used FLT3 ligand-cultured mouse bone marrow-derived DCs (FL-DCs) and chimeric HTLV-1. Phenotypically, the FL-DCs upregulated expression of surface markers (CD80, CD86, and MHC class II) on infection; however, the level of MHC class I remained unchanged. We performed kinetic studies to understand viral entry, proviral integration, and expression of the viral protein Tax. Multiplex cytokine profiling revealed production of an array of proinflammatory cytokines and type 1 IFN (IFN-α) by FL-DCs treated with virus. Virus-matured FL-DCs stimulated proliferation of autologous CD3(+) T cells as shown by intracellular nuclear Ki67 staining and produced IFN-γ when cultured with infected FL-DCs. Gene expression studies using type 1 IFN-specific and DC-specific arrays revealed upregulation of IFN-stimulated genes, most cytokines, and transcription factors, but a distinct downregulation of many chemokines. Overall, these results highlight the critical early responses generated by FL-DCs on challenge with cell-free chimeric HTLV-1.

Depletion of dendritic cells enhances susceptibility to cell-free infection of human T cell leukemia virus type 1 in CD11c-diphtheria toxin receptor transgenic mice.

Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. The genesis of these diseases is believed to be associated with the route (mucosa versus blood) and mode (cell-free versus cell-associated) of primary infection as well as the modulation of dendritic cell (DC) functions. To explore the role of DCs during early HTLV-1 infection in vivo, we used a chimeric HTLV-1 with a replaced envelope gene from Moloney murine leukemia virus to allow HTLV-1 to fuse with murine cells, which are generally not susceptible to infection with human retroviruses. We also used a CD11c-diphtheria toxin receptor transgenic mouse model system that permits conditional transient depletion of CD11c(+) DCs. We infected these transgenic mice with HTLV-1 using both cell-free and cell-associated infection routes in the absence and presence of DCs. The ablation of DCs led to an enhanced susceptibility to infection with cell-free but not cell-associated HTLV-1 in both CD4 and non-CD4 fractions, as measured by the proviral load. Infection with cell-free virus in the absence of DCs was also found to have increased levels of Tax mRNA in the non-CD4 fraction. Moreover, depletion of DCs significantly dampened the cellular immune response (IFN-gamma(+)CD8(+) T cells) against both cell-free and cell-associated virus. These results uniquely differentiate the involvement of DCs in early cell-free versus late cell-associated infection of HTLV-1 and highlight a significant aspect of viral immunopathogenesis related to the progression of adult T cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis after the initial infection.

Cellulose acetate 1,2-benzenedicarboxylate inhibits infection by cell-free and cell-associated primary HIV-1 isolates.

Cellulose acetate 1,2-benzenedicarboxylate (CAP), a pharmaceutical excipient used for enteric film coating of capsules and tablets, was previously shown to have potent inhibitory activity against infection by human immunodeficiency virus type 1 (HIV-1) T cell line-adapted (TCLA) strains. In the present study, we determined the inhibitory activity of CAP against infection by cell-free and cell-associated primary HIV-1 isolates with distinct genotypes and biotypes in cervical explants, peripheral blood mononuclear cells (PBMCs), monocytederived macrophages (MDMs), and CEMx174 5.25M7 cells. CAP blocked infection by cell-free and cell-associated HIV-1 in cervical explants. It inhibited infection by cell-free primary HIV-1 isolates (clades A to G and group O) in PBMCs, MDMs, and CEMx174 5.25M7 cells and blocked transmissions of the cell-associated primary HIV-1 isolates from dendritic cells (DCs) to PBMCs, from MDMs to PBMCs, and from PBMCs to CEMx174 5.25M7 cells. The inhibitory activity of CAP on infection by the cell-free and cell-associated primary HIV-1 isolates is independent of viral subtypes and coreceptor usage. These data suggest that CAP is a good microbicide candidate that can be further developed for preventing sexual transmission of HIV-1.

Proteinase-activated receptor-2 induction by neuroinflammation prevents neuronal death during HIV infection.

Proteinase-activated receptors (PARs), a newly discovered subgroup of G-protein coupled receptors, are widely expressed by neural cells, but their roles in the nervous system remain uncertain. In this study, we report that PAR-2 was up-regulated on neurons in conjunction with neuroinflammation in brain tissue from patients with HIV-1-associated dementia. The inflammatory cytokines TNF-alpha and IL-1beta were also increased in HIV-1-associated dementia brains compared with patients without dementia (p < 0.05), but these same cytokines induced PAR-2 expression on neurons. Enhanced PAR-2 expression and subsequent activation prevented neuronal cell death and induction of the tumor suppressor, p53, caused by the HIV-encoded protein, Tat (p < 0.01). Intrastriatal implantation of a PAR-2 peptide agonist also inhibited Tat-induced neurotoxicity in a mouse model of HIV neuropathogenesis (p < 0.05). Moreover, PAR-2 null animals showed more severe neuroinflammation and neuronal loss caused by Tat neurotoxicity (p < 0.05). TNF-alpha protected wild-type neurons from Tat-related neurotoxicity, but in PAR-2-deficient neurons, the same concentrations of TNF-alpha were cytotoxic (p < 0.001). Thus, neuroinflammation can exert protective effects by which it induces PAR-2 expression with the ensuing abrogation of neuronal death.

Enzymatic characterization of membrane-associated hepatitis C virus NS3-4A heterocomplex serine protease activity expressed in human cells.

The hepatitis C virus (HCV) nonstructural (NS)3-NS4A serine protease heterocomplex is a prime target for development of novel HCV therapies, due to its essential role in maturation of the viral polyprotein. While the mode of substrate/inhibitor recognition of the HCV NS3/NS4A serine protease has been extensively studied in vitro, important molecular aspects of the mechanism of action for this membrane-bound multifunctional enzyme remain unresolved in vivo. In particular, what influence does membrane association exert on the specificity and catalysis of NS3-4A protease? To carry out this study, we developed a specific and sensitive protease assay using a unique internally quenched fluorogenic substrate (IQFS). Our IQFS enables for the first time the direct, specific detection of NS3-4A protease activity within membrane fractions isolated from human cells expressing NS3-4A and the determination of its steady-state kinetic parameters, which were found to be K(m) = 51 +/- 3 microM and k(cat) = 0.39 min(-1). We also show that our fluorescence-based bioassay can be used to evaluate specifically the potency and mode of action of NS3-4A directed inhibitors, such as in the case of a known NS3-4A substrate-analogue inhibitor (K(i) = 22 nM). Our results indicate that the membrane anchoring of NS3 by NS4A does not affect the substrate/inhibitor recognition by the NS3-4A protease domain. Further investigation may reveal whether membrane association could be important for regulating other enzymatic activities associated with NS3 (e.g., helicase and/or ATPase) and/or regulating the recently proposed cross-talk between the protease and helicase activities.

Comparing capsid assembly of primate lentiviruses and hepatitis B virus using cell-free systems.

Many viruses that assemble their capsids in the eukaryotic cytoplasm require a threshold concentration of capsid protein to achieve capsid assembly. Strategies for achieving this include maintaining high levels of capsid protein synthesis and targeting to specific sites to raise the effective concentration of capsid polypeptides. To understand how different viruses achieve the threshold capsid protein concentration required for assembly, we used cell-free systems to compare capsid assembly of hepatitis B virus (HBV) and three primate lentiviruses. Capsid formation of these diverse viruses in a common eukaryotic extract was dependent on capsid protein concentration. HBV capsid assembly was also dependent on the presence of intact membrane surfaces. Surprisingly, not all of the primate lentiviral capsid proteins examined required myristoylation and intact membranes for assembly, even though all contain a myristoylation signal. These findings reveal significant diversity in how different capsid proteins assemble in the same cellular extract.

The viral protein 3CD induces an equilibrium between the viral protein and RNA synthesis in a cell-free system for poliovirus replication.

In a cell-free system derived from uninfected HeLa cells and programmed with poliovirus RNA, an unbalance between the different replication steps is observed. After programming, the vRNA is exclusively used as a template for viral translation. It takes hours before there is a switch from protein synthesis to RNA replication. This is probably the reason for the inefficient production of infectious virus (compared to the synthesis in infected cells). If, however, the cell-free system is programmed with vRNA and with a mRNA coding for the viral protein 3CD, an increase in vRNA synthesis is found early post-programming, resulting in a better balance of protein synthesis and RNA synthesis and an increased virus yield of at least 2 log10. These data show that a balance between translation, RNA replication and packaging is required to allow efficient viral proliferation. The virus yield could be increased by a further log10 by the addition of pirodavir (a capsid stabiliser) and 10% of rabbit reticulocyte lysate to the cell-free system.

Bleomycin-induced alterations in DNA replication: relationship to DNA damage.

Bleomycin (BLM), a well-known DNA scission agent, is assumed to inhibit intracellular DNA replication by damaging the DNA template (cis-acting mechanism), although other DNA damaging compounds can alter DNA replication through modulation of crucial replication factor(s) (trans-acting mechanism). The present study examines the relationship between DNA damage and inhibition of replication caused by BLM in the well-defined simian virus 40 (SV40) intracellular and cell-free in vitro systems. Treatment of SV40-infected BSC-1 cells for 2 h with BLM at 50 microg/mL, induced 0.3 break/viral genome. Under the same treatment conditions, analysis of replication intermediates on two-dimensional gels showed a decrease in both mass of SV40 replication intermediates and replication activity. The mass of SV40 intermediates was decreased to about 30%, whereas replication activity was reduced to less than 5%. These results suggest that BLM inhibits both initiation and elongation phases of SV40 replication. In a cell-free DNA replication system, extracts from BLM-treated cells (50 micro/mL) were able to support SV40 DNA replication by only 50%. In this study, non-drug-treated DNA template was used, implying that BLM can induce a trans-acting effect. Finally, the drug-induced effects on SV40 DNA replication in cell-free and intracellular viral systems were compared to the effects on genomic DNA replication in BSC-1 cells. Overall, the results support the concept that BLM-induced inhibition of DNA replication occurs by both trans- (inhibition of replication of nondamaged template) and cis-acting mechanisms (template damage).

Direct immunosuppressive effects of EBV-encoded latent membrane protein 1.

In neoplastic cells of EBV-positive lymphoid malignancies latent membrane protein (LMP1) is expressed. Because no adequate cellular immune response can be detected against LMP1, we investigated whether LMP1 had a direct effect on T lymphocyte activation. In this study we show that nanogram amounts of purified recombinant LMP1 (rLMP1) strongly suppresses activation of T cells. By sequence alignment two sequences (LALLFWL and LLLLAL) in the first transmembrane domain of LMP1 were identified showing strong homology to the immunosuppressive domain (LDLLFL) of the retrovirus-encoded transmembrane protein p15E. The effects of rLMP1 and LMP1-derived peptides were tested in T cell proliferation and NK cytotoxicity assays and an Ag-induced IFN-gamma release enzyme-linked immunospot assay. LMP1 derived LALLFWL peptides showed strong inhibition of T cell proliferation and NK cytotoxicity, while acetylated LALLFWL peptides had an even stronger effect. In addition, Ag-specific IFN-gamma release was severely inhibited. To exert immunosuppressive effects in vivo, LMP1 has to be excreted from the cells. Indeed, LMP1 was detected in supernatant of EBV-positive B cell lines (LCL), and differential centrifugation in combination with Western blot analysis of the pellets indicated that LMP1 is probably secreted by LCL in the form of exosomes. The amount of secreted LMP1 in B cell cultures is well below the immunosuppressive level observed with rLMP1. Our results demonstrate direct immunosuppressive properties of LMP1 (fragments) and suggest that EBV-positive tumor cells may actively secrete LMP1 and thus mediate immunosuppressive effects on tumor-infiltrating lymphocytes. Moreover, we demonstrate, for the first time, that transmembrane protein-mediated immunosuppression is not solely restricted to RNA tumor viruses, but can also be found in DNA tumor viruses.

Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-gamma-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy.

We have constructed a recombinant defective adenovirus that expresses functional murine IFN-gamma-inducible protein-10 (IP-10) chemokine (AdCMVIP-10). Injection of AdCMVIP-10 into s.c. tumor nodules derived from the CT26 murine colorectal adenocarcinoma cell line displayed some antitumor activity but it was not curative in most cases. Previous studies have shown that injection of similar s. c. CT26 tumor nodules with adenovirus-encoding IL-12 (AdCMVIL-12) induces tumor regression in nearly 70% of cases in association with generation of antitumor CTL activity. AdCMVIP-10 synergizes with the antitumor effect of suboptimal doses of AdCMVIL-12, reaching 100% of tumor eradication not only against injected, but also against distant noninjected tumor nodules. Colocalization of both adenoviruses at the same tumor nodule was required for the local and distant therapeutic effects. Importantly, intratumoral gene transfer with IL-12 and IP-10 generated a powerful tumor-specific CTL response in a synergistic fashion, while both CD4 and CD8 T cells appeared in the infiltrate of regressing tumors. Moreover, the antitumor activity of IP-10 plus IL-12 combined gene therapy was greatly diminished by simultaneous in vivo depletion of CD4+ and CD8+ T cells but was largely unaffected by single depletion of each T cell subset. An important role for NK cells was also suggested by asialo GM1 depletion experiments. From a clinical point of view, the effects of IP-10 permit one to lower the required gene transfer level of IL-12, thus preventing dose-dependent IL-12-mediated toxicity while improving the therapeutic efficacy of the elicited antitumor response.

Murine cytomegalovirus infection down-regulates MHC class II expression on macrophages by induction of IL-10.

Herpesviruses utilize many strategies for weakening the host immune response. For CMV, this includes avoidance of NK clearance and inhibition of MHC class I and class II presentation pathways. In this study, we report that mouse CMV (MCMV) specifically causes a premature and transient activation of host IL-10 very early in the course of infection, resulting in a dramatic and selective reduction in MHC class II surface expression. The expression of IL-10 is normally late in the immune response to a pathogen, serving to dampen the response by suppression of the production of inflammatory cytokines. In infection of macrophages, we show that MCMV induces the production of IL-10, leading to an early and selective reduction in the expression of MHC class II on the surface of the cells. Inhibition of MHC class II expression was not observed in the presence of neutralizing Abs to IL-10 or in macrophages from IL-10-deficient mice. Moreover, MCMV-infected IL-10-deficient mice developed an early and significantly more robust macrophage MHC class II induction than normal mice. Altogether, our results demonstrate that viral induction of an IL-10 autocrine pathway plays an essential early role in selectively reducing MHC class II expression on the surface of APC prior to stimulation by IFN-gamma.

Controlling human immunodeficiency virus type 1 gene expression by unnatural peptides.

Small unnatural peptides that target specific RNA structures have the potential to control biological processes. RNA-protein interactions are important in many cellular functions, including transcription, RNA splicing, and translation. One example of such interactions is the mechanism of trans-activation of human immunodeficiency virus type 1 (HIV-1) gene expression that requires the interaction of Tat protein with the trans-activation responsive region (TAR) RNA, a 59-base stem-loop structure located at the 5'-end of all nascent HIV-1 transcripts. We report here a synthetic peptide derived from Tat sequence (37-72), containing all D-amino acids, that binds in the major groove of TAR RNA and interferes with transcriptional activation by Tat protein in vitro and in HeLa cells. Our results indicate that unnatural peptides can inhibit the transcription of specific genes regulated by RNA-protein interactions.

Bypass of a site-specific cis-Syn thymine dimer in an SV40 vector during in vitro replication by HeLa and XPV cell-free extracts.

The key step in skin cancer induction by UV light is thought to be the mutagenic DNA synthesis past a DNA photoproduct in a proto-oncogene or tumor suppressor gene. To investigate this critical step, we have constructed an SV40 vector containing a cis-syn thymine dimer, the major DNA photoproduct induced by UVB light, within an AseI site at a location that would initially be replicated by leading strand synthesis. When the dimer-containing SV40 vector was incubated with cell-free HeLa extracts in the presence of TAg, and then digested with AseI, a 2325 bp fragment corresponding to inhibition of cleavage at the dimer site was observed, suggesting that the dimer had terminated synthesis and/or had been bypassed. When the reaction was limited to one round of replication and the products of restriction enzyme digestion were examined by denaturing gel electrophoresis, bands corresponding to both termination and bypass were observed in roughly a one-to-one ratio. Whereas increasing the dNTP concentration from 10 microM to 1 mM increased the ratio of bypass to termination from 0.6 to 2.6, it had no effect on the site of termination, which occurred exclusively one nucleotide before the dimer. Experiments in which dGTP was held constant at 25 microM and various combinations of the remaining nucleotides were raised from 25 microM to 1 mM showed substantial increases in the bypass-to-termination ratio, with the greatest effect seen for raising all three nucleotides to 1 mM. Replication by primary fibroblast XPV extracts was also investigated and found to be greatly stimulated by rhRPA, whereas the stimulatory effect for HeLa cell extracts was variable. In the presence of rhRPA, the XPV extracts were also found to bypass the cis-syn dimer, which contrasts with a recent report that could not detect dimer bypass in SV40 transformed XPV extracts in the absence of added replication factors [Cordeiro-Stone, M., et al. (1997) J. Biol. Chem. 272, 13945-13954].

Efficient transduction of hemopoietic CD34+ progenitors of human origin using an original retroviral vector derived from Fr-MuLV-FB29: in vitro assessment.

A novel retroviral vector has been designed based on a Friend-murine leukemia virus (Fr-MuLV) FB29 strain. The latter has been selected according to characteristics of pathogenicity in mice where it induces a disease of the haemopoietic system affecting all lineages. Higher infectivity has also been demonstrated as compared to other strains. In accordance with these findings, the amphotropic producer clone used in this study carrying along the neomycine resistance gene (FOCH-Neo), harbors viral titers over 10(7) cfu/ml. To investigate the potential of genetically engineering hematopoietic precursors, CD34+ progenitors were selected from cord blood, bone marrow, and peripheral blood mobilized stem cells (patients + solid tumors) and transduced with FOCH-Neo. High transduction rates were achieved using virus supernatant and minimal doses of hematopoietic growth factors during pretransduction and transduction steps. A polymerase chain reaction (PCR) assay investigating the presence of both neomycin-encoding and viral vector sequences tested positive in 45-90% of granulocyte-macrophage colony-forming units (CFU-GM) generating cells (bone marrow and peripheral blood derived cells) following transduction. An average of 35% colonies showed resistance to G418. Such levels of transduction proved reproducible using only supernatants harboring over 10(7) cfu/ml. In those experiments where long-term in vitro cultures could be maintained over 5 weeks (all cord blood and 5 among 23 PBSC), efficient transduction of long-term culture initiating cell (LTC-IC) hematopoietic progenitors was demonstrated on the basis of both resistance to G418 and virus integration. In the latter case, the PCR assay tested positive in as much as 35-60% of late unselected CFU-colonies. This novel retroviral vector harbors interesting features toward genetic modification of hematopoietic progenitors.

Virus-mediated release of endosomal content in vitro: different behavior of adenovirus and rhinovirus serotype 2.

Endosomal penetration by nonenveloped viruses might be accomplished by either local breakdown of the endosomal membrane (e.g., adenovirus) or formation of a membrane-spanning pore by capsid proteins. Uncoating of the nonenveloped virus human rhinovirus serotype 2 (HRV2) has been shown to occur from late endosomes and to be entirely dependent on the acidic pH in this compartment (Prchla, E., E. Kuechler, D. Blaas, and R. Fuchs. 1994. J. Virol. 68: 3713-3723). To investigate further the mechanism of uncoating of HRV2, an in vitro assay was established to test viruses or virus-derived peptides for their capacity to release cointernalized biotin-dextran of different molecular mass (10 and 70 kD) from isolated endosomes. The suitability of the assay was demonstrated by use of a fusogenic peptide derived from influenza virus hemagglutinin (GALA-INF3). Whereas adenovirus induced a low pH-dependent release of up to 46% of the internalized biotin-dextran and did not show any significant size selectivity (as expected for endosome disruption), HRV2 mediated release of 27% of the 10 kD dextran and only traces of the 70-kD dextran. Similarly, GALA-INF3-induced release of biotin-dextran was also size dependent. The potential role of the capsid protein VP1 in HRV2 uncoating in vivo was also substantiated in our in vitro system using an amphipathic, NH2-terminal peptide of VP1. Taken together, these data favor the model of a specific pore-forming mechanism for HRV2 uncoating which is in contrast to the membrane-disrupting mechanism of adenovirus.

SV40 VP1 assembles into disulfide-linked postpentameric complexes in cell-free lysates.

The simian virus 40 (SV40) capsid is composed of pentameric capsomeres of the major structural protein, Vp1. The chemical nature of Vp1-Vp1 interactions, as well as the role of the minor structural proteins, Vp2 and Vp3, in SV40 assembly is not clear. We show here that Vp1 molecules synthesized in rabbit reticulocyte lysates self-assembled into postpentameric 12S complexes in the absence of other viral structural proteins and in a time and concentration dependent manner. The 12S complexes were resistant to perturbants of noncovalent interactions but were sensitive to reduction by dithiothretiol. Nonreducing SDS-PAGE analysis revealed disulfide-linked VP1 complexes of > 400 kDa. Our results are consistent with crystallography studies of SV40 which suggest involvement of disulfide bonds at a postcapsomeric stage of viral assembly.