Increase in infectivity of targeted Moloney murine leukemia virus-based gene-delivery vectors through lowering the threshold for fusion.

Many research groups have developed targeted vectors for gene therapy based on Moloney murine leukemia virus (MoMLV). Despite proper binding of the targeted vector to the target molecule, little or no infectivity of human cells expressing the target molecule has been achieved in most studies. One of the reasons for this lack of infectivity may be steric hindrance within the targeted envelope glycoprotein (Env), impeding the conformational changes required for fusion and infection. Here, attempts were made to solve this problem by mutating key residues within Env of two targeted MoMLV-based vectors, MoMLV-E-Sel and MoMLV-FBP. Selection of key residues was based on an Env with reduced threshold for fusion, that of the CD4-independent human immunodeficiency virus type 2 isolate ROD/B. It was shown here that vectors bearing MoMLV-FBP Env with a V512M substitution had higher titres and faster kinetics of entry than vectors bearing parental targeted Env proteins. This could be due to the partial release of steric constraints that result in an Env with a reduced threshold for fusion.

Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry.

Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-beta-cyclodextrin (MbetaC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway.

Evidence for nonspecific adsorption of targeted retrovirus vector particles to cells.

The ability to specifically target a cell-type is important for the development of vectors for in vivo gene therapy. In order to produce retrovirus vectors targeting ovarian cancer cells, which specifically overexpress alpha folate receptor (alphaFR), a single chain antibody was fused as an N-terminal extension of the ecotropic and amphotropic murine leukemia virus (MLV) envelope glycoproteins. Vector particles bearing the modified glycoproteins were produced and analysed. Although conventional FACS studies indicated that viral particles bearing the modified Env could bind to ovarian cancer cells, targeted infection was not achieved. The initial step of virus-cell interaction was further studied using an immunofluorescence technique, which allows visualisation of single retrovirus particles. Vectors bearing chimeric or wild-type glycoproteins bound equally well to cells with or without the targeted receptor, although soluble chimeric glycoproteins bound specifically to FBP. Our results indicate that the incorporation of specific ligands to the virus envelope does not necessarily result in significant enhancement of vector particle binding. A similar interaction was also observed using Env-defective virus particles, suggesting that cellular factors incorporated into the lipid envelope play a dominant role in promoting initial adsorption of virus particles to cells. Significant implications arise from these observations on the interpretation of previous reports on 'targeted' vectors, and for the development of vectors for in vivo gene therapy protocols.

Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors.

To date, only adherent cell lines have been used for the generation of packaging cells for the production of type C retrovirus vectors. The large-scale production of high titre retrovirus vectors could benefit from the development of packaging cells growing in suspension. Here, we describe the ability of two different lymphoid cell lines, one B- and one T-lymphoblastoid cell line (Namalwa and CEM, respectively), to produce MLV-based vectors. Upon transfection with a third generation packaging construct, the virus particle production by Namalwa cells was characterised by low RT-activity, and by CEM cells as high RT activity as previously established adherent packaging cells. An amphotropic packaging cell line (CEMFLYA) was therefore established from CEM cells. Upon introduction of a lacZ vector genome, the novel packaging cell line produced vector particles routinely in the region of 10(7) infectious units/ml. The vectors were helper-free and highly stable in fresh human serum. The potential for scaled up vector production was demonstrated by continuous culture of the new packaging cells for 14 days in a 250 ml spinner flask. These suspension packaging cells should be applicable to large bioreactor systems to bulk produce high titre, complement-resistant retrovirus vectors for gene therapy.

HIV type 1 protease cleavage site mutations and viral fitness: implications for drug susceptibility phenotyping assays.

The recombinant virus assay (RVA) is a method for assessing the susceptibility of human immunodeficiency virus type 1 (HIV-1) plasma isolates to antiretroviral drugs. The RVA involves the production of viable virus in vitro by homologous recombination of RT-PCR products from plasma virus with a noninfectious reverse transcriptase (RT) or protease (PR)-deleted cloned HIV-1 provirus. In this study, we have constructed RVA plasmids with contiguous deletions in RT, PR, and the p7/p1 and p1/6 gag protease cleavage sites (CS). The deletions in these plasmids allow generation of recombinant viruses with all loci currently identified as important for resistance to anti-HIV-1 drugs being derived from the clinical isolate, including CS mutations that compensate for the reduced fitness of viruses resistant to protease inhibitors (Doyon et al., J Virol 1996:70:3763-3769). We have also used these new constructs to generate viruses with or without compensatory CS mutations, and examined the effects on fitness. In the case of an indinavir-selected virus, fitness was restored close to that of a wild type virus when a vector deleted in the CS and PR was used. With an amprenavir-selected isolate, virus fitness was incompletely restored by including the CS, and this defect appeared to be partially due to reduced infectivity of the virions. We conclude that the CS mutations were required for optimum detection of resistance in the RVA, but that virus fitness can remain compromised even in the presence of compensatory CS mutations.

Lung surfactant protein A provides a route of entry for respiratory syncytial virus into host cells.

Lung surfactant protein A (SP-A) has a central role in host defense mediated by the interaction of surface carbohydrates of inhaled pathogens with the lectin domains of SP-A. Respiratory syncytial virus (RSV), the most important viral pathogen of neonates and infants, encodes a highly glycosylated attachment protein, G. Binding studies were performed with G-protein from RSV (human, A2 strain) and human SP-A. The effect of SP-A on the interaction between RSV and host cells was determined by two methods: an infectivity study with monolayers of Hep-2C cells and by interleukin-8 (IL-8) release from buffy coat (BC) cells. SP-A binds to RSV G-protein in a concentration-dependent manner that is inhibitable by both ethylenediamine tetraacetic acid (EDTA) and mannan, indicating that binding is through the carbohydrate recognition domain of the SP-A and a carbohydrate moiety of the G-protein. The level of RSV infection of Hep-2C cells increases with increasing concentrations of SP-A. The amount of IL-8 released by BC cells in the presence of RSV is increased with SP-A concentrations of 2.9 microg/mL or greater. Our results show that SP-A enhances the attachment of RSV and subsequent entry into host cells. The effect of SP-A on viral uptake by epithelial cells and macrophage may determine both innate and adaptive immune responses to RSV.

Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction.

The initial step of virus-cell interaction was studied by immunofluorescence microscopy. Single particles of murine leukemia virus (MLV) vectors and human immunodeficiency virus (HIV) were visualized by immunofluorescence. Fluorescent dots representing single virions could be localized by staining of capsid proteins (CA) or surface envelope proteins (SU) after fixation of virus supernatants. This technique can be used to determine particle concentration in viral supernatants and also to study virus-cell interaction. We investigated the role of the Env-receptor interaction for the initial binding event between the cell and the viral particles. Ecotropic MLV vector particles were shown to bind to human cells which do not express the specific viral receptor. In addition, MLV particles defective for Env were shown to bind the cells similarly to infectious MLV. Time course experiments of virus-cell binding and dissociation showed identical profiles for infectious and Env-defective MLV particles and suggested that MLV Env is not involved in the early phases of attachment of virus to cells. The possible implication of cellular factors in enhancing viral binding and infectivity is discussed.

Human immunodeficiency virus. Mutations in the viral protease that confer resistance to saquinavir increase the dissociation rate constant of the protease-saquinavir complex.

Mutations in the human immunodeficiency virus (HIV) protease (L90M, G48V, and L90M/G48V) arise when HIV is passaged in the presence of the HIV protease inhibitor saquinavir. These mutations yield a virus with less sensitivity to the drug (L90M > G48V >> L90M/G48V). L90M, G48V, and L90M/G48V proteases have 1/20, 1/160, and 1/1000 the affinity for saquinavir compared to WT protease, respectively. Therefore, the affinity of mutant protease for saquinavir decreased as the sensitivity of the virus to saquinavir decreased. Association rate constants for WT and mutant proteases with saquinavir were similar, ranging from 2 to 4 x 10(7) M-1 s-1. In contrast, the dissociation rate constants for WT, L90M, G48V, and L90M/G48V proteases complexed with saquinavir were 0.0014, 0.019, 0.128, and 0. 54 s-1, respectively. This indicated that the reduced affinity for mutant proteases and saquinavir is primarily the result of larger dissociation rate constants. The increased dissociation rate constants may be the result of a decrease in the internal equilibrium between the bound inhibitor with the protease flaps up and the bound inhibitor with the flaps down. Interestingly, the affinity of these mutant proteases for VX-478, ABT-538, AG-1343, or L-735,524 was not reduced as much as that for saquinavir. Finally, the catalytic constants of WT and mutant proteases were determined for eight small peptide substrates that mimic the viral cleavage sites in vivo. WT and L90M proteases had similar catalytic constants for these substrates. In contrast, G48V and L90M/G48V proteases had catalytic efficiency (kcat/Km) values with TLNF-PISP, RKIL-FLDG, and AETF-YVDG that were 1/10 to 1/20 the value of WT protease. The decreased catalytic efficiencies were primarily the result of increased Km values. Thus, mutations in the protease decrease the affinity of the enzyme for saquinavir and the catalytic efficiency with peptide substrates.

Expression of TAR RNA-Binding Protein in Baculovirus and Co-Immunoprecipitation with Insect Cell Protein Kinase.

TAR RNA-binding protein TRBP was originally isolated by its binding affinity for radiolabeled HIV-1 leader RNA. Subsequent studies have suggested that this protein is one member of a family of double-stranded RNA-binding proteins. Recent findings indicate that TRBP might function to antagonize the translational inhibitory effect that can be mediated through cellular protein kinase, PKR. Here, we report on the over-expression of a cDNA coding for TRBP in eukaryotic SF9 cells using baculovirus. We characterized the nuclear localization of TRBP in insect cells, and we demonstrate that TRBP co-immunoprecipitates with a protein in these cells antigenically related to human PKR. Copyright 1995 S. Karger AG, Basel

Analysis of resistance to human immunodeficiency virus type 1 protease inhibitors by using matched bacterial expression and proviral infection vectors.

There are already reports, from clinical trials with human immunodeficiency virus type 1 protease inhibitors, of the emergence of drug-resistant mutants which have one or more point mutations in their protease genes. To examine roles of individual and multiple amino acid substitutions in terms of altered enzyme and virus drug sensitivities, we have produced matched vectors for bacterial expression and virus production. Both vectors accept the same restriction enzyme fragment, produced by PCR or PCR-mutagenesis of the protease gene, allowing parallel expression of mutant enzymes in Escherichia coli and in recombinant viruses. The utility of this vector system was demonstrated by using protease variants glycine to valine at amino acid 48 (G48V) and leucine to methionine at amino acid 90 (L90M) identified after passage of HIV-1 in the Roche phase II clinical trial protease inhibitor Ro 31-8959 (H. Jacobsen, K. Yasargil, D. L. Winslow, J. C. Craig, A. Krohn, I. B. Duncan, and J. Mous, Virology 206:527, 1995). G48V, L90M, and G48V/L90M exhibited successively less processing in vitro than the wild-type enzyme, and the purified enzymes were 220-, 20-, and 720-fold, respectively, less sensitive to Ro 31-8959. The reduced enzyme sensitivity correlated directly with the sensitivities of the matched recombinant viruses, in that individual mutations L90M and G48V conferred 2-fold and 4- to 6-fold increases in 50% inhibitory concentration, respectively, whereas G48V/L90M was 8 to 10 times less sensitive to Ro 31-8959. A proviral vector with the entire protease gene deleted was constructed for use as an in vivo recombination target for an overlapping protease PCR fragment, generating wild-type infectious virus. Finally, direct ligation of restriction fragments, generated from random PCR mutagenesis, into the proviral vector should provide a library of protease mutations that allow extremely rapid selection of highly resistant viral variants.

Cross-resistance analysis of human immunodeficiency virus type 1 variants individually selected for resistance to five different protease inhibitors.

Human immunodeficiency virus type 1 (HIV-1) protease inhibitor-resistant variants, isolated on passage of HIV-1HXB2 in MT-4 cells with five different protease inhibitors, have been examined for cross-resistance to five inhibitors. The protease inhibitors studied were Ro 31-8959, A-77003, XM323, L-735,524, and VX-478. Resistant variants with two to four mutations within their protease sequence and 9- to 40-fold-decreased susceptibility were selected for all five inhibitors within six to eight passes in cell culture. Passage of a zidovudine-resistant mutant in Ro 31-8959 generated a dual reverse transcriptase- and protease-resistant virus. Variants were cloned directly into a modified pHXB2-D infectious clone for cross-resistance analysis. Although the resistant variants selected possessed different combinations of protease mutations for each inhibitor, many showed cross-resistance to the other inhibitors, and one showed cross-resistance to all five inhibitors. Interestingly, some mutants showed increased susceptibility to some inhibitors. Further HIV passage studies in the combined presence of two protease inhibitors demonstrated that in vitro it was possible to delay significantly selection of mutations producing resistance to one or both inhibitors. These studies indicate that there may be some rationale for combining different protease inhibitors as well as protease and reverse transcriptase inhibitors in HIV combination therapy.

A Simple and Rapid TAR-Dependent in vitro Transcription Assay Using T Cell Nuclear Extracts and Synthetic tat1-86 Protein.

A 'G-less' cassette was cloned downstream of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) to facilitate rapid identification of authentic LTR-directed transcription in T cell nuclear extracts. Despite a high constitutive level of transcription, addition of chemically synthesized full-length HIV-1(bru) tat (amino acids 1-86) stimulated transcription 3-fold but only if the template included the TAR region of the LTR. Suppression of basal transcription in T cell nuclear extracts by sodium citrate increased the relative level of tat stimulation, but neither potassium chloride nor histone H1 had an effect. A mutant synthetic tat polypeptide, lacking residues 22-32 in the cysteine-rich domain, was inactive in uptake assays and failed to stimulate transcription. Short basic peptides that competed full-length tat from complexes with TAR RNA also inhibited tat stimulation of transcription, whereas short basic peptide unable to bind TAR or compete tat from complexes were also unable to inhibit tat stimulation of transcription. These data confirm that active HIV-1 tat must first interact with TAR RNA via basic amino acid residues in order to stimulate transcription of downstream sequences. Copyright 1995 S. Karger AG, Basel

A bioactive fullerene peptide.

The highly hydrophobic C60 (buckminsterfullerene) was water solubilized by covalently linking the synthon 1,2-dihydro-1,2-methanofullerene [60]-61-carboxylic acid to the alpha-amino group of the hydrophilic 4-8 sequence of peptide T, known to display potent human monocyte chemotaxis. The resulting compound, characterized by a variety of analytical techniques, including a UV spectrum in aqueous solution, exhibits remarkable chemotactic potency, comparable to that of the parent pentapeptide. Furthermore, this fullerene-peptide conjugate inhibits, albeit weakly, HIV-1 protease.

Relationship of age and sex-role conflict for professional women in human services.

This study provides external validation of the Sex-role Conflict Scale. The relationship between age and sex-role conflict among women with educational interests was investigated. 128 female students in education were assessed on the conflict they experienced in various settings. The mean sex-role conflict scores, although hypothesized to be significantly different, were not among women ages 25 yr. and below, ages 26 to 39 yr., and ages 40 yr. and over. Also investigated was the relationship between age and sex-role conflict in dual-career respondents. Of the 29 respondents who met the criterion for classification, mean sex-role conflict scores were not significantly different for the three age groups. The nonlinear relationship between sex-role conflict and age does not have support for professional women who work in fields other than business.

Transcriptional activation is not responsible for increased levels of autonomously expressed simian virus 40 T-antigen in herpes simplex virus-infected cells.

Herpes simplex virus type 1 (HSV-1) superinfection of CV-1 cells weakly transactivated a plasmid-borne metallothionein 1 (MT-1) promoter, but activated the expression of a marker gene controlled by an authentic HSV-1 promoter to a high level. In contrast, CMT-3 cells, which are CV-1 cells stably transformed with the simian virus 40 (SV40) large T-antigen (T-Ag) gene controlled by the MT-1 promoter, contained high levels of T-Ag following HSV-1 superinfection, but only if cells were preincubated in the presence of heavy-metal ions. This T-Ag was functional in that it could mediate the increase in copy number of a marker plasmid containing the SV40 origin of DNA replication. Pulse and continuous labeling of preinduced CMT-3 cells showed that T-Ag expression was not induced by HSV-1; but rather, HSV-1 superinfection resulted in the stabilization of pre-existing protein.

Transcriptional activation with concurrent or nonconcurrent template replication has differential effects on transient expression from herpes simplex virus promoters.

We have used two methods to induce template replication in order to assess the effect on expression of marker genes controlled by herpes simplex virus type 1 (HSV-1) promoters. One method used the HSV-1 origin of DNA replication from the short repeat region of the viral genome (HSV-1 oris), and allowed simultaneous replication and transcriptional activation of the plasmid-borne template. The other, using the simian virus 40 origin of replication (SV40 ori) allowed plasmid template replication prior to activation of transcription by HSV-1 infection. The two regimes had markedly different effects upon the levels of reporter gene activity induced by HSV-1 superinfection. Replication of reporter plasmids using the SV40 ori yielded levels of reporter gene activity proportional to plasmid copy number when cells were superinfected with HSV-1. In contrast, our results indicated that sequences containing, or in close proximity to, the HSV-1 oris in the reporter plasmid had a significant inhibitory effect on expression from all viral promoters whether or not the plasmid was allowed to replicate. Still, the early (beta) promoter-controlled reporter enzyme activity declined at late times while that controlled by the strict late (gamma) promoter was significantly higher following HSV-1 oris-mediated template replication.

Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability.

To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation.

Organization of terminal reiterations in the virion DNA of herpesvirus saimiri.

The population of herpesvirus saimiri (HVS) genomes extracted from extracellular virions are double-stranded, linear DNA molecules of about 160 kilobase pairs (kbp) each composed of a central segment of 110 to 112 kbp and 36% (G + C) (i.e. 'light' or L-DNA) linked to direct reiterations of a 1.44 kbp repeat unit of 71% (G + C) (i.e. 'heavy' or H-DNA) at each terminus. In this paper, we show that the population of HVS DNA molecules contains approximately equal concentrations of genomes with all possible integral numbers of complete repeat units (i.e. from greater than 30 to 1) at either 'left' or 'right' ends but that all molecular ends are derived by a unique cleavage at a site close to the single ApaI restriction endonuclease site of the H-DNA repeat unit. Junctions of proximal H-DNA repeat units with L-DNA occur at, or very close to, the sequence present at the molecular ends. The transition from L- to H-DNA occurs abruptly at this site at the 'right' end of the L-DNA component but some rearranged restriction enzyme cleavage sites typical of H-DNA are found within the first 0.8 kbp of the L-DNA sequences at the 'left' H-L DNA junction. HVS appears to provide an extreme example of the general process whereby herpesvirus DNAs are matured from concatemeric intermediates by a site-specific cleavage/recombination process involving random choice between equivalent sites for the initiation of the process and with choices between alternative termination sites being limited by a headful packaging mechanism.

A single regulatory region modulates both cis activation and trans activation of the herpes simplex virus VP5 promoter in transient-expression assays in vivo.

A detailed analysis of the expression of the bacterial chloramphenicol acetyltransferase gene controlled by the herpes simplex virus major capsid protein (VP5) promoter showed that this promoter can be functionally separated into an 80-base core region, which has the minimal information required to serve as a pol II promoter but which is not fully activated by viral superinfection or by cotransfections with plasmids bearing functional alpha (immediate-early) genes, and an approximately 100-base regulatory region upstream of the core, which allowed full induction of VP5 promoter-driven chloramphenicol acetyltransferase activity but which repressed the ability of the VP5 core promoter to be cis activated by the simian virus 40 enhancer. This was in distinct contrast to the situation with the alkaline exonuclease promoter (a model early promoter) and defined the regions of this promoter which can be used to study the interaction between viral promoters and putative regulatory proteins induced by viral infection.

Evidence that the major delayed-early DNA-binding proteins of herpesvirus saimiri are bound to DNA in vivo.

Associations of herpesvirus saimiri-specified proteins with nuclear fractions from cultures of infected cells were probed by nuclease digestion, detergent extractions and extractions and immunofluorescence microscopy using monoclonal antibodies to virus polypeptides. Nuclease digestion selectively released delayed-early polypeptides with apparent mol. wt. of 110 000 (110K) and 51 000 (51K) from nuclei of infected cultures and the majority of each of these polypeptides partitioned with the insoluble fraction after detergent extraction of such nuclei. However, the nuclease-mediated release of both these proteins was specifically reduced when nuclei were isolated from cultures in which virus DNA synthesis had been inhibited with phosphonoacetic acid (PAA). In addition, the 110K polypeptide partitioned into the soluble fraction when nuclei from PAA-treated cultures were extracted with detergent. Immunofluorescence microscopy revealed characteristic and distinctive subnuclear localizations of the 110K and 51K polypeptides in control cultures and these patterns of subnuclear accumulations were markedly altered in cultures treated with PAA. We conclude that the DNA-binding properties of the delayed-early 110K and 51K proteins of herpesvirus saimiri previously observed in vitro are likely to reflect their functions as DNA-binding proteins in vivo.