Retroviral packaging cell line

The present invention provides packaging cell lines and recombinant lentiviral or retroviral particles produced therefrom, particularly pseudotyped retroviral particles. The packaging cell lines of the invention are produced by inducibly expressing an envelope protein by methods described herein. Also described is a screening assay for compounds that affect integration of viral nucleic acid into target (e.g., host) nucleic acid. Such compounds are identified based on their effect on viral integrase.

FIELD OF THE INVENTION
 This invention relates generally to the field of recombinant retroviral
 particles for use in gene delivery and more specifically to packaging cell
 lines for producing retroviral particles.
 BACKGROUND OF THE INVENTION
 Retroviruses are enveloped RNA viruses that, after infection of a host
 cell, reverse transcribe their RNA genomes into a DNA intermediate, or
 provirus. All viruses containing an RNA genome and producing an
 RNA-dependent DNA polymerase are contained in the retroviral family. The
 family is divided into three subfamilies: (1) Oncovirinae, including all
 the oncogenic retroviruses, and several closely related non-oncogenic
 viruses; (2) Lentivirinae, the "slow retroviruses" such as the human
 immunodeficiency virus (HIV) and visna virus; and (3) Spumavirinae, the
 "foamy" retroviruses that induce persistent infections, generally without
 causing any clinical disease. Retroviruses contain at least three types of
 proteins encoded by the viral genome, i.e., gag proteins (the group
 antigen internal structural proteins), pol proteins (the RNA-dependent DNA
 polymerase and the protease and integrase proteins), and env proteins (the
 viral envelope protein or proteins). In addition to genes encoding the
 gag, pol, and env proteins, the genome to the retrovirus includes two long
 terminal repeat (LTR) sequences, one at the 5' and one at the 3' end of
 the virus. These 5' and 3' LTRs promote transcription and polyadenylation
 of viral mRNAs and participate in the integration of the viral genome into
 the cellular DNA of the host.
 The provirus can be stably integrated into the host's cellular DNA. Gene
 products encoded by the provirus are then expressed by the host cell to
 produce retroviral virions, thereby replicating the virus. Because the
 retroviral genome can be manipulated to include exogenous nucleotide
 sequence(s) of interest for expression in a target cell, retroviral
 vectors are important tools for stable gene transfer into mammalian cells.
 Many proposed gene therapy applications use retroviral vectors to take
 advantage of the ability of these naturally infectious agents to transfer
 and efficiently express recombinant nucleotide sequences in susceptible
 target cells. Retroviral vectors suitable for use in such applications are
 generally defective retroviral vectors that are capable of infecting the
 target cell, reverse transcribing their RNA genomes, and integrating the
 reverse transcribed DNA into the target cell genome, but are incapable of
 replicating within the target cell to produce infectious retroviral
 particles (e.g., the retroviral genome transferred into the target cell is
 defective in gag, and/or in pol, see Coffin, J., In: RNA Tumor Viruses,
 Weiss, R. et al. (ed) Cold Spring Harbor Laboratory, Vol. 2, pp. 36-73,
 1985).
 Retroviral vectors and packaging cells (helper cells) have been developed
 to introduce recombinant nucleic acid molecules into mammalian cells
 without the danger of the production of replicating infectious virus. This
 methodology uses two components, a retroviral vector and a packaging cell.
 The retroviral vector contains long terminal repeats (LTRs), the foreign
 DNA to be transferred, and a packaging sequence. This retroviral vector
 will not reproduce by itself because the genes which encode the structural
 and envelope proteins are not included within the vector. The packaging
 cell contains genes encoding the gag, pol, and env proteins, but does not
 contain the packaging signal, so that the cell can only form empty virus
 particles by itself. With this method, the retroviral vector is introduced
 into the packaging cell, to create a cell able to produce virus. The cell
 manufactures viral particles containing only the retroviral vector DNA,
 and therefore has been considered safe.
 Use of retroviral vectors is limited in many aspects. For example, although
 retroviruses can efficiently infect and stably integrate into the genome
 of rapidly-dividing cells, retroviral integration into the genome of
 non-dividing or slowly dividing cells is inefficient (Springett et al.
 1989 J. Virol. 63:3865-3869; Miller et al. 1990 Mol. Cell. Biol.
 10:4239-4242; Roe et al. 1993 EMBO J. 12:2099-2108). Most packaging
 systems provide only modest vector titers, and the fragility of retroviral
 vector particles complicate purification and concentration (Paul et al.
 1993 Hum. Gene Therap. 4:609-615). Finally, retroviruses enter target
 cells by binding of retroviral envelope glycoproteins (encoded by the env
 gene) to specific target cell surface receptors. This envelope
 protein-cell surface receptor interaction is often species specific, and
 in some cases even tissue specific. Moreover, the level of expression of
 the cell surface receptor on the target cells can vary widely among target
 cells. As a result, retroviruses usually have a limited host range
 (Kavanaugh et al. 1994 Proc. Natl. Acad. Sci. USA 91:7071-7075; Hopkins
 1993 Proc. Natl. Acad Sci. USA 90:8759-8760).
 One strategy for both expanding retroviral host cell range and increasing
 the structural stability of the retroviral virion involves production of
 pseudotyped retroviral viral vectors. Pseudotyped retroviral vectors
 useful in transformation of target cells are generally composed of
 retroviral virion structural proteins (e.g, gag proteins), a recombinant
 RNA genome containing the nucleotide sequence of interest, the pol protein
 for reverse transcription of the recombinant RNA contained in the virion,
 and a non-retroviral envelope protein or an envelope protein from a
 different retrovirus. The recombinant RNA genome is usually replication
 defective, e.g., defective in the pol and/or gag genes, to prevent
 production of infectious retrovirus following transfer of the nucleotide
 sequence of interest into the target cell. The envelope protein of the
 pseudotyped retrovirus is normally selected to provide a broader host
 range or to provide selective targeting of cells to be infected.
 SUMMARY OF THE INVENTION
 The present invention provides packaging cell lines and recombinant
 retroviral particles produced therefrom, particularly pseudotyped
 retroviral particles. Exemplary packaging cell lines are derived from 293
 HeLa, Cf2Th, D17, MDCK, or BHK cells, most preferably from 293 cells.
 Retroviral particles are produced by inducibly expressing an envelope
 protein of interest (e.g., a retroviral envelope or the envelope protein
 of vesicular stomatitis virus (VSV G)). Inducible expression of the
 envelope protein is accomplished by operably linking an envelope
 protein-encoding nucleotide sequence to an inducible promoter (e.g., a
 promoter composed of a minimal promoter sinked to at least one copy of
 tetO, the binding site for the tetracycline repressor (tetR) of the
 Escherichia coli tetracycline resistance operon Tn10). Expression from the
 inducible promoter is regulated by a transactivating factor, composed of a
 first ligand-binding domain that negatively regulates transcription from
 the inducible promoter (e.g., a prokaryotic tetracycline repressor
 polypeptide (tet R)). Transcription of the envelope-encoding nucleotide
 sequence under control of the inducible promoter is activated by a
 transactivator when tetracycline is absent.
 A primary object of the invention is to provide a packaging cell line that
 produces retroviral particles, especially pseudotyped retroviral
 particles, more efficiently (e.g., at higher titers) than conventional
 packaging cell lines. The packaging cell line of the invention is
 characterized by a first polynucleotide having an HIV genome operably
 linked to a first inducible promoter wherein the HIV genome is defective
 for cis-acting elements, for self-replication and for expression of
 functional Env protein; a second polynucleotide encoding a functional
 heterologous Env protein operably linked to a second inducible promoter;
 and a third polynucleotide encoding a regulatable transcriptional
 activator controlling transcription from the first and second inducible
 promoters.
 Another object of the invention is to provide recombinant retroviral
 vectors, especially pseudotyped retroviral vectors, that are useful in
 both in vitro and in vivo transformation of a target cell.
 An advantage of the invention is that packaging cell lines can be produced
 that contain an envelope-encoding nucleotide sequence stably integrated in
 the cell's genome, which sequence can be inducibly expressed, thus
 allowing generation of packaging cell lines capable of expressing an
 envelope protein that is otherwise toxic to the host cell (e.g., VSV G).
 Another advantage of the invention is that the packaging cell lines do not
 have the potential to produce replication competent retroviruses.
 In another embodiment, the invention provides a method for producing a
 packaging cell line. The method includes transfecting a suitable cell with
 i) a first polynucleotide having an HIV genome operably linked to a first
 inducible promoter W-herein the HIV genome is defective for cis-acting
 elements, for self-replication and for expression of functional Env
 protein; ii) a second polynucleotide encoding a functional heterologous
 Env protein operably linked to a second inducible promoter; and iii) a
 third polynucleotide encoding a regulatable transcriptional activator
 controlling transcription from the first and second inducible promoters;
 and culturing the transfected cells above under conditions which allow
 screening for cells containing proteins encoded by the polynucleotides.
 Preferably, the cell is co-transfected with a selectable marker encoding
 polynucleotide, such as an antibiotic resistance gene.
 In another embodiment, the invention provides a method of detecting a viral
 nucleic acid sequence integrated into a target nucleic acid sequence by
 reversibly immobilizing the target nucleic acid sequence to a solid
 support, contacting the target nucleic acid with a viral preintegration
 complex (PIC) containing the viral nucleic acid sequence, under such
 conditions and for sufficient time so as to allow the viral nucleic acid
 sequence to integrate into the target nucleic acid sequence and detecting
 the integrated viral nucleic acid sequence.
 In yet another embodiment, the invention provides a method of detecting
 viral integrase activity contained in viral preintegration complex (PIC).
 The method includes reversibly immobilizing a target nucleic acid sequence
 to a solid support; contacting the target nucleic acid with the viral
 preintegration complex (PIC) containing a viral nucleic acid sequence,
 under such conditions and for sufficient time so as to allow the viral
 nucleic acid sequence to integrate into the target nucleic acid sequence
 and detecting the integrated viral nucleic acid sequence. The presence of
 the viral nucleic acid sequence in the target nucleic acid sequence is
 indicative of integrase activity.
 In a further embodiment, the invention provides a method of identifying a
 compound that affects viral integrase activity contained in a
 preintegration complex (PIC). The method includes reversibly immobilizing
 a target nucleic acid sequence to a solid support; contacting the target
 nucleic acid with the viral preintegration complex (PIC) containing a
 viral nucleic acid sequence and a test compound suspected of affecting
 integrase activity, under such conditions and for sufficient time so as to
 allow the viral nucleic acid sequence to integrate into the target nucleic
 acid sequence; and detecting the integrated viral nucleic acid sequence.
 The amount of viral nucleic acid sequence integrated in the target nucleic
 acid sequence is indicative of the effect of the compound on integrase
 activity.
 These and other objects, advantages and features of the present invention
 will become apparent to those persons skilled in the art upon reading the
 details of the vectors, cell lines and methodology as more fully set forth
 below.

DESCRIPTION OF THE PREFERRED EMBODIMENTS
 Before the present inducible expression system, use of the inducible
 expression system to generate packaging cell lines for retroviral vectors
 pseudotyped with VSV G, and constructs, vector particles, and packaging
 cell lines associated therewith are described, it is to be understood that
 this invention is not limited to the particular methodology, protocols,
 cell lines, retroviruses, vectors, constructs, and reagents described as
 such may, of course, vary. It is also to be understood that the
 terminology used herein is for the purpose of describing particular
 embodiments only, and is not intended to limit the scope of the present
 invention which will be limited only by the appended claims.
 It must be noted that as used herein and in the appended claims, the
 singular forms "a", "and", and "the" include plural referents unless the
 context clearly dictates otherwise. Thus, for example, reference to "a
 packaging cell" includes a plurality of such cells and reference to "the
 retroviral vector" includes reference to one or more vectors and
 equivalents thereof known to those skilled in the art, and so forth.
 Unless defined otherwise, all technical and scientific terms used herein
 have the same meaning as commonly understood to one of ordinary skill in
 the art to which this invention belongs. Although any methods, devices and
 materials similar or equivalent to those described herein can be used in
 the practice or testing of the invention, the preferred methods, devices
 and materials are now described.
 All publications mentioned herein are incorporated herein by reference for
 the purpose of describing and disclosing the cell lines, vectors, and
 methodologies which are described in the publications which might be used
 in connection with the presently described invention. The publications
 discussed above and throughout the text are provided solely for their
 disclosure prior to the filing date of the present application. Nothing
 herein is to be construed as an admission that the inventors are not
 entitled to antedate such disclosure by virtue of prior invention.
 Definitions
 By "inducible expression system" is meant a construct or combination of
 constructs that includes a nucleotide sequence encoding a transactivator,
 an inducible promoter that can be transcriptionally activated by the
 transactivator, and a nucleotide sequence of interest operably linked to
 the inducible promoter. For example, an exemplary inducible expression
 system of the invention includes a nucleotide sequence encoding a
 tetracycline operon regulatable transactivator (tTA) and a nucleotide
 sequence of interest operably linked to an inducible promoter composed of
 a minimal promoter operably linked to at least one tetO sequence.
 By "transactivator," "transactivating factor," or "transcriptional
 activator" is meant a polypeptide that facilitates transcription from a
 promoter. Where the promoter is an inducible promoter, the transactivator
 activates transcription in response to a specific transcriptional signal
 or set of transcriptional signals. For example, in the inducible
 expression system of the invention, tTA is a transactivator that
 facilitates transcription from the inducible tetO promoter when tTA is not
 bound to tetracycline.
 "Tetracycline repressor protein," "tetracycline repressor polypeptide,"
 "tetR polypeptide," and "tetR protein" are used interchangeably herein to
 mean a polypeptide that exhibits both 1) specific binding to tetracycline
 and/or tetracycline derivatives; and 2) specific binding to tetO sequences
 when the tetR polypeptide is not bound by tetracycline or a tetracycline
 analog(s). "TetR polypeptide" is meant to include a naturally-occurring
 (i.e., native) tetR polypeptide sequence and functional derivatives
 thereof.
 By "transcriptional activation domain" is meant a polypeptide sequence that
 facilitates transcriptional activation from a promoter. "Transcriptional
 activation domain" includes transcriptional activation domains derived
 from the naturally-occurring amino acid sequence of a transcription factor
 as well as functional derivatives thereof.
 By "envelope protein" is meant a polypeptide that 1) can be incorporated
 into an envelope of a retrovirus; and 2) can bind target cells and
 facilitate infection of the target cell by the RNA virus that it envelops.
 "Envelope protein" is meant to include naturally-occurring (i.e., native)
 envelope proteins and functional derivatives thereof that 1) can form
 pseudotyped retroviral virions according to the invention, and 2) exhibit
 a desired functional characteristic(s) (e.g, facilitate viral infection of
 a desired target cell, and/or exhibit a different or additional biological
 activity). In general, envelope proteins of interest in the invention
 include any viral envelope protein that can, in combination with a
 retroviral genome, retroviral Pol, retroviral Gag, and other essential
 retroviral components, form a retroviral particle. Such envelope proteins
 include retroviral envelope proteins derived from any suitable retrovirus
 (e.g., an amphotropic, xenotropic, ecotropic or polytropic retrovirus) as
 well as non-retroviral envelope proteins that can form pseudotyped
 retroviral virions (e.g., VSV G). Envelope proteins of particular interest
 include, but are not limited to, envelope protein of vesicular stomatis
 virus (VSV G), HTLV-1, gibbon ape leukemia virus (GALV), Sindai virus,
 influenza virus, herpes virus, rhabdovirus, and rabies virus.
 By "functional derivative of a polypeptide" is meant an amino acid sequence
 derived from a naturally-occurring polypeptide that is altered relative to
 the naturally-occurring polypeptide by virtue of addition, deletion,
 substitution, or other modification of the amino acid sequence.
 "Functional derivatives" contemplated herein exhibit the characteristics
 of the naturally-occurring polypeptide essential to the operation of the
 invention. For example, by "functional derivative of tetR" is meant a
 polypeptide derived from tetR that retains both 1) tetracycline or
 tetracycline analog binding and 2) the ability to inhibit transcriptional
 activation by tTA when bound to tetracycline or an analog thereof.
 By "promoter" is meant a minimal DNA sequence sufficient to direct
 transcription of a DNA sequence to which it is operably linked. The term
 "promoter" is also meant to encompass those promoter elements sufficient
 for promoter-dependent gene expression controllable for cell-type specific
 expression, tissue-specific expression, or inducible by external signals
 or agents; such elements may be located in the 5' or 3' regions of the
 naturally-occurring gene.
 By "inducible promoter" is meant a promoter that is transcriptionally
 active when bound to a transcriptional activator, which in turn is
 activated under a specific condition(s), e.g., in the presence of a
 particular chemical signal or combination of chemical signals that affect
 binding of the transcriptional activator to the inducible promoter and/or
 affect function of the transcriptional activator itself. For example, the
 transcriptional activator of the present invention, tTA, induces
 transcription from its corresponding inducible promoter when tetracycline
 is absent, i.e. tetracycline is not bound to tTA.
 By "construct" is meant a recombinant nucleotide sequence, generally a
 recombinant DNA molecule, that has been generated for the purpose of the
 expression of a specific nucleotide sequence(s), or is to be used in the
 construction of other recombinant nucleotide sequences. In general,
 "construct" is used herein to refer to a recombinant DNA molecule.
 By "operably linked" is meant that a DNA sequence and a regulatory
 sequence(s) are connected in such a way as to permit gene expression when
 the appropriate molecules (e.g., transcriptional activator proteins) are
 bound to the regulatory sequence(s).
 By "operatively inserted" is meant that a nucleotide sequence of interest
 is positioned adjacent a nucleotide sequence that directs transcription
 and translation of the introduced nucleotide sequence of interest (i.e.,
 facilitates the production of, e.g., a polypeptide encoded by a DNA of
 interest).
 By "packaging cell line" is meant a line of packaging cells selected for
 their ability to package defective retroviral vectors at a titer of
 generally greater than 10.sup.3 virions per milliliter of tissue culture
 medium, having less than 10 helper virus virions per milliliter of tissue
 culture medium, and capable of being passaged in tissue culture without
 losing their ability to package defective retroviral vectors.
 By "transformation" is meant a permanent or transient genetic change,
 preferably a permanent genetic change, induced in a cell following
 incorporation of new DNA (i.e., DNA exogenous to the cell). Where the cell
 is a mammalian cell, a permanent genetic change is generally achieved by
 introduction of the DNA into the genome of the cell.
 By "target cell" is meant a cell(s) that is to be transformed using the
 methods and compositions of the invention. Transformation may be designed
 to non-selectively or selectively transform the target cell(s). In
 general, target cell as used herein means a eukaryotic cell that can be
 infected by a VSV G pseudotyped retroviral vector according to the
 invention.
 By "transformed cell" is meant a cell into which (or into an ancestor of
 which) has been introduced, by means of recombinant DNA techniques, a DNA
 molecule encoding a gene product (e.g., RNA and/or protein) of interest
 (e.g., nucleic acid encoding a therapeutic cellular product).
 By "nucleotide sequence of interest" or "DNA of interest" is meant any
 nucleotide or DNA sequence that encodes a protein or other molecule that
 is desirable for expression in a host cell (e.g., for production of the
 protein or other biological molecule (e.g., a therapeutic cellular
 product) in the target cell). The nucleotide sequence of interest is
 generally operatively linked to other sequences which are needed for its
 expression, e.g., a promoter. In general, a nucleotide sequence of
 interest present in the genome of a recombinant retroviral particle of the
 invention encodes any gene product of interest, usually a therapeutic gene
 product where the recombinant retroviral particle is to be used to
 transform cells in vivo (e.g., in a gene therapy application in humans).
 By "therapeutic gene product" is meant a polypeptide, RNA molecule or other
 gene product that, when expressed in a target cell, provides a desired
 therapeutic effect, e.g., repair of a genetic defect in the target cell
 genome (e.g., by complementation), expression of a polypeptide having a
 desired biological activity, and/or expression of an RNA molecule for
 antisense therapy (e.g., regulation of expression of a endogenous or
 heterologous gene in the target cell genome).
 By "subject" or "patient" is meant any subject for which cell
 transformation or gene therapy is desired, including humans, cattle, dogs,
 cats, guinea pigs, rabbits, mice, insects, horses, chickens, and any other
 genus or species having cells that can be infected with a viral vector
 having an envelope containing VSV G or other envelope described herein.
 By "transgenic organism" is meant a non-human organism (e.g., single-cell
 organisms (e.g., yeast), mammal, non-mammal (e.g., nematode or
 Drosophila)) having a non-endogenous (i.e., heterologous) nucleic acid
 sequence present as an extrachromosomal element in a portion of its cells
 or stably integrated into its germ line DNA.
 By "transgenic animal" is meant a non-human animal, usually a mammal,
 having a non-endogenous (i.e., heterologous) nucleic acid sequence present
 as an extrachromosomal element in a portion of its cells or stably
 integrated into its germ line DNA (i.e., in the genomic sequence of most
 or all of its cells). Heterologous nucleic acid is introduced into the
 germ line of such transgenic animals by genetic manipulation of, for
 example, embryos or embryonic stem cells of the host animal.
 By "viral vector" is meant a recombinant viral particle that accomplishes
 transformation of a target cell with a nucleotide sequence of interest.
 By "virion," "viral particle," or "retroviral particle" is meant a single
 virus minimally composed of an RNA genome, Pol protein (for reverse
 transcription of the RNA genome following infection), Gag protein
 (structural protein present in the nucleocapsid), and an envelope protein.
 As used herein, the RNA genome of the retroviral particle is usually a
 recombinant RNA genome, e.g., contains an RNA sequence exogenous to the
 native retroviral genome and/or is defective in an endogenous retroviral
 sequence (e.g., is defective in pol, gag, and/or env, and, as used herein,
 is normally defective in all three genes).
 By "pseudotyped viral particle," or "pseudotyped retroviral particle" is
 meant a viral particle having an envelope protein that is from a virus
 other than the virus from which the RNA genome is derived. The envelope
 protein can be from a retrovirus of a species different from the
 retrovirus from which the RNA genome is derived or from a non-retroviral
 virus (e.g., vesicular stomatitis virus (VSV)). Preferably, the envelope
 protein of the pseudotyped retroviral particle is VSV G.
 By "VSV G" or "VSV G envelope protein" is meant the envelope protein of
 vesicular stomatitis virus (VSV) or a polypeptide derived therefrom or
 recombinant fusion polypeptide having a VSV G polypeptide sequence fused
 to a heterologous polypeptide sequence, where the VSV G-derived
 polypeptide of recombinant fusion polypeptide can be contained in a viral
 envelope of a pseudotyped retroviral particle and retains infectivity for
 a desired target cell (e.g., a range of desired eukaryotic cells, or a
 specific target cell of interest).
 By "VSV G pseudotyped virus," "VSV G pseudotyped retrovirus," "VSV G
 pseudotyped viral particle," or "VSV G pseudotyped retroviral particle,"
 is meant a retrovirus having the envelope protein VSV G, e.g., either in
 combination with or substantially substituted for the endogenous
 retroviral envelope. Preferably, VSV G is present in the VSV G pseudotyped
 viral envelope such that VSV G represents about 50% of the envelope
 protein(s) present in the envelope, more preferably about 75%, even more
 preferably about 90% to about 95%, still more preferably greater than
 about 95%, most preferably about 100% or such that VSV G is substantially
 the only envelope protein present in the pseudotyped viral particle
 envelope.
 The invention will now be described in further detail.
 Packaging Cell Line and Transactivator
 In a first embodiment, the invention provides a packaging cell line which
 includes a first polynucleotide having an HIV genome operably linked to a
 first inducible promoter wherein the HIV genome is defective for
 cis-acting elements, for self-replication and for expression of functional
 Env protein; a second polynucleotide encoding a functional heterologous
 Env protein operably linked to a second inducible promoter; and a third
 polynucleotide encoding a regulatable transcriptional activator
 controlling transcription from the first and second inducible promoters.
 The first polynucleotide includes an HIV genome that is incapable of
 self-replication and encodes gag and pol but not env, and does not contain
 a packaging signal or long-terminal repeats (LTRs). The second
 polynucleotide encodes a heterologous Env or "envelope protein" as
 described above in the definitions.
 The third polynucleotide encodes a regulatable transcriptional activator,
 or transactivator, which controls transcription from the first and second
 inducible promoters, which regulate expression of the first and second
 polynucleotides described above.
 In a preferred embodiment, the first, second and third polynucleotides are
 contained in vectors. These polynucleotides can be contained in one or
 more vectors, preferably plasmid vectors. In the exemplary packaging cell
 line of the invention, the first polynucleotide is contained in a first
 plasmid vector referred to pPTK and the second polynucleotide is contained
 in second plasmid vector referred to as pBIGFVG. The second vector
 contains the VSV envelope as well as green fluorescent protein as an
 indicator marker. The third polynucleotide encoding a regulatable
 transcriptional activator is exemplified herein as containing a minimal
 CMV immediate-early gene promoter linked to seven tandem copies of the
 tetR-binding site replaced the CMV promoter(BglII/BamHI fragment), in
 pcDNAneo to create phCMVn. As discussed herein, other viral envelopes and
 other indicator markers will be known to those of skill in the art for use
 in the present invention.
 In one aspect of the invention, one or more polynucleotides encoding
 retroviral accessory proteins, are included as part of the first or second
 polynucleotide constructs, for example. Accessory proteins include vpr,
 vif, nef, vpx, tat, rev, and vpu
 Preferably, the transcriptional activator, or transactivator can be
 expressed at high levels in a eukaryotic cell without significantly
 adversely affecting general cellular transcription in the host cell
 transactivator expression that is sufficient to facilitate transactivation
 of the inducible promoter, but that is not detrimental to the cell (e.g.,
 is not toxic to the cell). "High levels" can be a level of expression that
 allows detection of the transactivator by Western blot. The transactivator
 can preferably be expressed in a wide variety of cell types, including
 mammalian and non-mammalian cells such as, but not limited to, human,
 monkey, mouse, hamster, cow, insect, fish, and frog cells.
 The transactivator can be expressed either in vivo or in vitro, and
 expression of the transactivator can be controlled through selection of
 the promoter to which the nucleotide sequence encoding the transactivator
 is operably linked. For example, the promoter can be a constitutive
 promoter or an inducible promoter. Examples of such promoters include the
 human cytomegalovirus promoter IE (Boshart et al., 1985 Cell 41:521-530),
 ubiquitously expressing promoters such as HSV-Tk (McKnight et al., 1984
 Cell 37:253-262) and .beta.-actin promoters (e.g. the human .beta.-actin
 promoter as described by Ng et al., Mol. Cell Biol. 1985 5:2720-2732).
 The promoter of the transactivator can be a cell type-specific or
 tissue-specific promoter that preferentially facilitates transcription of
 the transactivator in a desired cell of tissue type. Exemplary cell
 type-specific and/or tissue-specific promoters include promoters such as
 albumin (liver specific; Pinkert et al., 1987 Genes Dev. 1:268-277),
 lymphoid specific promoters (Calame et al., 1988 Adv. Immunol.
 43:235-275); in particular promoters of T-cell receptors (Winoto et al.,
 1989 EMBO J. 8:729-733) and immunoglobulins; Banerji et al., 1983 Cell
 33729--740; Queen and Baltimore, ibid. 741-748), neuron-specific promoters
 (e.g. the neurofilament promoter; Byrne et al., 1989 Proc. Natl. Acad.
 Sci. U.S.A. 86:5473-5477), pancreas-specific promoters (Edlunch et al.,
 1985 Science 230:912-916) or mammary gland-specific promoters (milk whey
 promoter, U.S. Pat. No. 4,873,316 and European Application Publication No.
 264, 166). Promoters for expression of the transactivator can also be
 developmentally-regulated promoters as the murine homeobox promoters
 (Kessel et al., 1990 Science 249:374-379) or the .alpha.-fetoprotein
 promoter (Campes et al., 1989 Genes Dev. 3:537-546). The promoter can be
 used in combination with control regions allowing integration site
 independent expression of the transactivator (Grosveld et al., 1987 Cell
 51:975-985). Preferably, the promoter is constitutive in the respective
 cell types. Preferably the promoter is a CMV promoter, more preferably a
 CMV immediate early gene promoter.
 Preferably, the transactivator is a native tetR polypeptide or a functional
 derivative of tetR, since tetR binds its specific DNA sequence (tetO) with
 greater affinity than either lacR or GAL4 bind their respective sequences.
 For example, tetR binds tetracycline much tighter
 (k.sub.a.apprxeq.10.sup.9 M.sup.-1 ; Takahashi et al., J Mol. Biol.
 187:341-348 (1986) than lacR complexes IPTG (k.sub.a.apprxeq.10.sup.6
 M.sup.-1 ; Barkley & Bourgeios in The Operon, Miller & Rezinkoff, eds.,
 Cold Spring Harbor Lab., Cold Spring Harbor, N.Y., 1980, pp. 177-220).
 Thus, very low, nontoxic concentrations of tetracycline function
 effectively. By "functional derivative of tetR" is meant a polypeptide
 derived from tetR that retains both 1) tetracycline or tetracycline analog
 binding and 2) the ability to inhibit transcription from the inducible
 promoter by preventing binding of the tetR domain to tetO sequences within
 the inducible promoter. The nucleotide sequence encoding tetR can be
 obtained according to Postel et al., 1984 Nucl. Acids Res. 2:4849-4863,
 herein incorporated by reference. Other tetR sequences useful in the
 invention, and the respective binding sites for these repressors, are
 described in (Waters et al., 1983 Nucl. Acids Res. 11:6089-6105; Postle et
 al., supra; Unger et al., 1984 Gene 31:103-108; Unger et al., 1984 Nucl.
 Acids Res. 127693--7703; Tovar et al., 1988 Mol. Gen. Genet. 215:76-80);
 for comparison and overview see Hillen and Wissmann in Protein-Nucleic
 Acid Interaction, Topics in Molecular and Structural Biology, Saenger and
 Heinemann (eds.), Macmillan, London, Vol. 10, pp. 143-162 (1989)).
 Tetracycline analogs can be any one of a number of compounds that are
 closely related to tetracycline and which bind to the tet repressor with a
 Ka of at least about 10.sup.6 M.sup.-1 (e.g., doxycycline). Preferably,
 the tetracycline analogs binds with an affinity of about 109 M-1 or
 greater, e.g., binds with an affinity of about 1011 M-1. Examples of such
 tetracycline analogs include, but re not limited to those disclosed by
 Hlavka and Boother, "The Tetracyclines," IN: Handbook of Experimental
 Pharmacology 78, R. K. Blackwood et al. (eds.), Springer-Verlag,
 Berlin-N.Y., 1985; Mitschef, "The Chemistry of the Tetracycline
 Antibiotics," Medicinal Research 9, Dekker, N.Y., 1978; Noyee Development
 Corporation, "Tetracycline Manufacturing Processes," Chemical Process
 Reviews, Park Ridge, N.J., 2 volumes, 1969; Evans, "The Technology of the
 Tetracyclines," Biochemical Reference Series 1, Quadrangle Press, New
 York, 1968; and Dowling, "Tetracycline," Antibiotics Monographs no. 3,
 Medical Encyclopedia, New York, 1955; each of which are incorporated
 herein by reference with respect to tetracycline analogs.
 Inducible Promoters
 In general, the inducible promoter used in conjunction with the
 transactivator in the inducible expression system of the invention is any
 promoter from which transcription can be regulated by the transactivator
 level.
 For example, where the transactivator is a tetR polypeptide, the inducible
 promoter is preferably a minimal promoter containing at least one tetO
 sequence, preferably at least 2 or more tandemly repeated tetO sequences,
 even more preferably at least 5 or more tandemly repeated tetO sequences,
 more preferably at least 7 tandemly repeated tetO sequences or more. The
 minimal promoter portion of the inducible promoter can be derived from any
 desired promoter, and is selected according to tet cell line in which the
 inducible expression system is to be used. Where the cell is a mammalian
 cell, a preferred minimal promoter is derived from CMV, preferably from
 the CMV immediate early gene 1A. In addition other inducible promoters
 could be employed, such as the ecdysone-inducible promoters (Invitrogen
 Inc., San Diego, Calif.) or the lacZ inducible promoters.
 Preferably, the inducible promoter is a minimal promoter operatively linked
 to at least one tet operator (tetO) sequence. The tetO sequence can be
 obtained, for example, according to Hillen & Wissmann 1989, supra, each of
 which are herein incorporated by reference with respect to the description
 and sequence of tetO. Other tetO sequences that can be used in the
 practice of the invention can be obtained from the following references:
 Waters et al., 1983, supra; Postle et al., 1984, supra; Unger et al.,
 1984, supra; Unger et al., 1984, supra; Tovar et al., 1988, supra; for
 comparison and overview see Hillen and Wissmann 1989, supra, the
 disclosures of which are fully herein incorporated by reference. One, two,
 three, four, five, six, seven, eight, nine or ten or more copies of the
 tet operator sequence can be used. Because multiple copies of the tet
 operator sequence provide a synergistic effect on the ability to control
 expression from these tetO-containing promoter, promoters having a greater
 number of copies of tetO allow an enhanced range of transactivator
 regulation of transcription from the promoter. Regulation of
 tetracycline-regulatable promoters containing tetO sequences is discussed
 in U.S. Pat. No. 5,464,758, and in Gossen and Brujand, 1992 Proc. Natl.
 Acad. Sci. U.S.A. 89:5547-5551, each of which are herein incorporated by
 reference.
 Constructs
 The basic components of the inducible expression system, e.g., the
 nucleotide sequence encoding the transactivator and the inducible promoter
 operably linked to a nucleotide sequence of interest, can be contained
 within a single "construct" or vector or within separate constructs. The
 construct can be derived from any of a variety of constructs know in the
 art and/or commercially available, and can be capable of replication in
 prokaryotic cells, eukaryotic cells, or, preferably, both prokaryotic and
 eukaryotic cells.
 In addition to the components described above, the construct can
 additionally contain nucleotide sequence encoding gene(s) that can serve
 as selectable markers, e.g., antibiotic resistance genes (e.g, ampicillin,
 hygromycin, G418), .beta.-galactosidase, or other gene products that can
 be used for selection of cells containing the construct. The construct can
 additionally contain other expression-facilitating sequences, such as
 enhancers, introns, or other sequences that facilitate expression of the
 transactivator and/or, where appropriate, expression of the nucleotide
 sequence of interest operably linked to the inducible promoter. In
 addition, indicators, such as green fluorescent protein (GFP) are useful
 in the constructs of the invention.
 Introduction of the Inducible Expression System Into Host Cells
 In another embodiment, the invention provides a method for producing a
 packaging cell line. The method includes transfecting a suitable cell, as
 described herein, with a first polynucleotide having an HW genome operably
 linked to a first inducible promoter wherein the HIV genome is defective
 for cis-acting elements, for self-replication and for expression of
 functional Env protein; a second polynucleotide encoding a functional
 heterologous Env protein operably linked to a second inducible promoter;
 and a third polynucleotide encoding a regulatable transcriptional
 activator controlling transcription from the first and second inducible
 promoters; and culturing the transfected cells of under conditions which
 allow screening for cells containing proteins encoded by the
 polynucleotides.
 Any eukaryotic cell line that can be stably transformed with the inducible
 expression system of the invention and the nucleotide sequence(s)
 expressing the essential retroviral components (e.g., Gag and Pol), and
 express sufficient levels of the essential retroviral components and
 inducibly express the desired envelope protein for the production of
 recombinant retroviral vectors according to the invention can be used to
 generate a packaging cell line according to the invention. Suitable host
 cells include cells of both mammalian (e.g., human, simian, canine,
 feline, equine, and rodent) and non-mammalian origin (e.g, insect,
 reptile, fish and avian). The packaging cells can be either an in vitro
 cell culture or present in an organism in vivo.
 Preferably, the packaging cells are present in an in vitro culture and can
 be cultured in a manner that allows for mass production of the recombinant
 retroviral vectors so as to provide suitable titers useful for production
 of the retroviral vector preparations for various applications (e.g., for
 clinical application in gene therapy). Where large-scale production of
 virus is desirable, the host cell from which the packaging cell is derived
 is preferably easy to culture, stable in long term culture (e.g., healthy
 cells can be maintained at relatively high cell densities for several days
 to several weeks or months, and do not undergo any significant genetic
 changes that might affect reliable production of consistent retroviral
 vector titers and/or the homogeneity of the genetic composition of the
 retroviral particles), and allows easy isolation of virus from the cell
 culture (e.g., by collection and concentration of cell culture supernatant
 to provide a crude retroviral particle preparation of an acceptable viral
 titer).
 As exemplified herein, the inducible expression system is introduced into a
 host cell as separate recombinant polynucleotide sequences (e.g., one
 encoding the transactivator and one encoding the gene product of interest
 under control of the inducible promoter). Introduction of the nucleotide
 sequence encoding the inducible expression system into host cells can be
 accomplished either in vitro or in vivo according to methods well known in
 the art (see, e.g., Sambrook et al., 1987 Molecular Cloning: A Laboratory
 Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.). In a
 preferred embodiment, the inducible expression system is introduced into
 the host cell by infection with a retroviral vector, the polynucleotide
 sequence encoding the transactivator and/or the inducible promoter
 operably linked to the polynucleotide sequence encoding the desired
 envelope protein, and the polynucleotide sequence encoding the inducible
 expression system is stably integrated into the host cell genome.
 Packaging Cell Lines Using the Inducible Expression System
 The inducible expression system of the invention is used to generate
 packaging cells useful in the production of recombinant pseudotyped
 retroviral vectors, which are suitable for use in the introduction of a
 DNA sequence of interest into a target cell. Pseudotyped retroviral
 particles are retroviral particles having an envelope protein that is
 derived from a virus other than the virus from which the viral RNA genome
 is derived. The envelope protein can be from a retrovirus of a species
 different from the retrovirus from which the RNA genome is derived or from
 a non-retroviral virus (e.g., vesicular stomatitis virus (VSV)). Normally,
 the pseudotyped lentiviral or retroviral vectors are defective, i.e., the
 retroviral vector is derived from a naturally-occurring virus that has
 been genetically altered to render the virus replication-defective. Once
 the virus delivers its genetic material into a target cell, the virus
 introduces the recombinant nucleotide sequence into cell, preferably as a
 stably chromosomally integrated sequence, but does not generate additional
 infectious virus upon expression of the introduced retroviral sequence.
 Alternatively, the retroviral vector containing the nucleotide sequence of
 interest is attenuated, i.e. does not cause significant pathology or
 morbidity in the infected host (i.e., the virus is nonpathogenic or causes
 only minor disease symptoms).
 Thus, in another embodiment of the invention, a method for producing a
 recombinant retrovirus which includes transfecting a packaging cell line
 as described herein with a lentiviral vector (e.g., a retroviral vector)
 including a polynucleotide encoding a packaging signal, an exogenous
 non-HIV polynucleotide, an RNA-export signal and lentiviral LTR sequences.
 A preferred RNA-export signal is a Rev response element.
 The exogenous non-HIV polynucleotide encodes a polypeptide which includes a
 growth factor, a cytokine, a hormone, a neurotrophic factor and an
 immunoregulatory agent. Such polypeptides are useful for gene therapy
 using a recombinant lentivirus produced by the method of the invention and
 include but are not limited to interleukins 1 through 15, interferons, and
 in particular gamma interferon (.gamma.-IFN), tumor necrosis factor (TNF),
 granulocyte-macrophage-colony stimulating factor (GM-CSF), and nerve
 growth factor (NGF).
 Pseudotyped lentiviral or retroviral particles can be produced by
 introducing a defective, recombinant lentiviral, or more specifically,
 retroviral genome into a packaging cell (e.g., by infection with a
 defective retroviral particle, or by other means for introducing DNA into
 a target cell (e.g., conventional transformation techniques)). The
 defective retroviral genome minimally contains the long terminal repeats,
 the exogenous nucleotide sequence of interest to be transferred, and a
 packaging sequence (.phi.). In general, the packaging cell provides the
 missing retroviral components essential for retroviral replication,
 integration, and encapsidation, and also expresses a nucleotide sequence
 encoding the desired envelope protein. However, the packaging cell does
 not have all of the components essential for the production of retroviral
 particles. The nucleotide sequence(s) encoding the missing viral
 component(s) in the packaging cell can be either stably integrated into
 the packaging cell genome, and/or can be provided by a co-infecting helper
 virus.
 The nucleotide sequences encoding the retroviral components and the
 lentiviral or retroviral RNA genome can be derived from any desired lenti-
 or retrovirus (e.g., murine, simian, avian, or human retroviruses). Most
 defective retroviruses developed for gene therapy applications are murine
 retroviruses (e.g., murine leukemia virus (MuLV), Moloney murine leukemia
 virus (MoMLV)), (see, e.g., Miller et al. 1992 Nature 357:455-460; and
 Mulligan 1993 Science 260:926-932). In general, the retroviral components
 can be derived from any retrovirus that can form pseudotyped retroviral
 particles with the desired envelope protein, e.g., VSV G. Where VSV G is
 the desired envelope protein, the retroviral components can be derived
 from MuLV, MoMLV, avian leukosis virus (ALV), human immunodeficiency virus
 (HIV), or any other retrovirus that can form pseudotyped virus with VSV G
 as the only envelope protein or with VSV G and a relatively small amount
 of retroviral envelope protein.
 In one example of a pseudotyped retrovirus produced according to the
 present invention, the free virion form of pseudotyped defective murine
 retrovirus contains the structural and enzymatic proteins of the
 retrovirus (including reverse transcriptase), two RNA copies of the
 retroviral genome, and portions of the cell's plasma membrane in which is
 embedded the desired viral envelope glycoprotein (e.g., VSV G). The genome
 is organized into four main regions: the long terminal repeat (LTR), the
 gag gene, the pol gene, and the env gene. The three genes gag, pol, and
 env, which are located between the terminal LTRs, encode internal viral
 structural proteins and reverse transcriptase, respectively, and the env
 gene encodes the envelope glycoprotein that confers infectivity and host
 range specificity to the virus. Preferably, the retroviral genome is
 defective in one or all three of these genes. In addition, the retroviral
 genome can contain a nucleotide sequence of interest to be ultimately
 transferred to a target cell. When the defective, recombinant retroviral
 genome is integrated into the host cell in its proviral form, the LTR is
 positioned at both ends of the proviral genome, and is a composite of the
 5' and 3' ends of the RNA genome. The LTR contains cis-acting elements
 necessary for the initiation and termination of transcription.
 An exemplary packaging cell of the invention contains genes encoding Gag
 and Pol, as well as the desired envelope protein, but does not contain the
 packaging signal ".phi." or the LTRs described above. Thus, a packaging
 cell can only form empty virion particles; once a retroviral RNA genome
 (which contains the nucleotide sequence of interest) is introduced into
 the packaging cell, the packaging cell can produce pseudotyped, defective
 retroviral particles. Packaging cells thus provide the missing retroviral
 components (i.e., the components for which the retroviral genome is
 defective) essential for viral replication in trans. Methods for
 production of replication-deficient retroviral genomes containing a
 nucleotide sequence of interest, as well as methods for generating a cell
 line expressing the gag and pol genes, are well known in the art and are
 described in, for example, U.S. Pat. No. 4,861,719; PCT published
 application no. WO 92/05266, published Apr. 2, 1992; and PCT published
 application no. WO 92/14829, published Sep. 2, 1992, each of which are
 incorporated herein by reference with respect to production of
 replication-deficient retroviral genomes and packaging cell lines
 expressing retroviral gag and pol genes. Retroviral packaging cell lines
 can be derived from any mammalian or non-mammalian cell that can express
 the retroviral Gag and Pol proteins, and can express the desired envelope
 protein (e.g., can tolerate expression of VSV G for several hours to
 several days, preferably for at least one week to two weeks or more).
 Preferably, the cell line from which the packaging cell line is derived is
 a cell selected from a liver, stroma, myogenic, fibroblast, and embryonic
 stem cell. Exemplary cells used by those of skill in the art for
 development of packaging cell lines include: 293 (ATCC CCL X) HeLa (ATCC
 CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10), or
 Cf2Th (ATCC CRL 1430) cell, most preferably a 293 cell, each of which are
 publicly available from the ATCC. The exemplary cell line of the invention
 is referred to as SODK1293 or SODK1.
 Pseudotyped retroviral particles are produced according to the invention by
 introducing a defective, recombinant retroviral genome containing a
 nucleotide sequence of interest into a packaging cell line that contains
 nucleotide sequences encoding for 1) functional retroviral proteins for
 which the introduced RNA genome is defective (e.g., gag and pol), and 2)
 an inducible expression system of the invention that facilitates
 expression of a desired envelope protein. The defective, recombinant RNA
 genome can be introduced into the packaging cell line by any means,
 including infection with a defective viral particle or other conventional
 means of transformation. Preferably, the packaging cell expresses a
 retroviral Gag protein, a retroviral Pol protein, and a desired envelope
 protein that is inducibly expressed using the system of the invention. The
 inducible expression system can be introduced as a single construct or as
 multiple constructs as described above and in the Examples.
 The inducible promoter is preferably a minimal promoter derived from a CMV
 early gene promoter which is operably linked to at least one tetO
 sequence, preferably at least 2 or more tandemly repeated tetO sequences.
 The nucleotide sequence encoding the desired envelope protein for
 production of retroviral particles is preferably a nucleotide sequence
 encoding a viral envelope protein derived from a virus other than the
 virus from which the Gag, Pol, and/or RNA genome are derived. Preferably,
 the viral envelope protein is derived from a retrovirus (e.g., a
 xenotropic retrovirus, a polytropic virus, an ecotropic or an amphotropic
 virus, preferably an amphotropic virus) or a non-retroviral virus, e.g., a
 rhabdovirus, Sindai virus, influenza virus, sindis virus, or herpes virus.
 In a preferred embodiment, the nucleotide sequence encoding the desired
 envelope protein encodes a rhabdovirus envelope G protein, such as the
 envelope protein of vesicular stomatitis virus (VSV), or a functional
 derivative thereof.
 The nucleotide sequence encoding VSV G is described in Rose et al. 1982
 Cell 30:753-762, herein incorporated by reference with respect to
 disclosure of the VSV G nucleotide and amino acid sequences. Where the
 desired envelope protein is VSV G, VSV G can be present as the only
 envelope protein in the pseudotyped retroviral virion, or can be present
 in combination with other envelope proteins (e.g., the retroviral envelope
 protein normally associated with the retrovirus from which the retroviral
 components of other pseudotyped virion are derived). Preferably, VSV G is
 present in the viral envelope such that VSV G represents about 50% of the
 envelope proteins present in the viral envelope, more preferably about
 75%, even more preferably about 90% to about 95%, still more preferably
 greater than 95%, most preferably about 100% or such that VSV G is
 substantially the only protein in the viral envelope. VSV G can be a
 native (i.e., naturally-occurring) VSV G, or a functional derivative
 thereof.
 Functional derivatives of VSV G include, but are not limited to, VSV
 G-derived polypeptides having amino acid substitutions, deletions,
 additions, and/or chemical modifications relative to native VSV G.
 Functional VSV G derivatives thus include, but are not limited to, VSV
 G-derived polypeptides having a function different from or in addition to
 that normally associated with native VSV G. For example, VSV G can be
 fused to a polypeptide derived from an antibody having binding affinity
 for a tissue-specific or cell-specific antigen. Pseudotyped viral
 particles having such a VSV G-single chain antibody fusion protein present
 in the viral envelope can preferentially infect cells expressing on their
 surface the antigen to which the antibody chain binds. Other VSV G
 functional derivatives can likewise alter the host cell range of the
 pseudotyped viral particle and/or provide other desired characteristics.
 In general, any VSV G functional derivative that can form pseudotyped
 retroviral virions according to the invention can be used.
 When the envelope-expressing packaging cell is exposed to tetracycline (or
 an analog thereof), substantially no or very little VSV G is expressed
 from the inducible promoter. In the absence of tetracycline, expression of
 the envelope protein is increased from about 10-fold to about 50-fold,
 preferably from about 40-fold to 90-fold, more preferably from about
 40-fold to 100-fold, and may be 200-fold or more relative to transcription
 in the presence of tetracycline. In addition, expression of the
 transactivator can be regulated by operably linking the nucleotide
 sequence encoding the transactivator to a regulatable promoter. Thus the
 packaging cell of the invention can provide two or more levels of
 regulation of expression of the desired envelope protein: 1) negative
 regulation of expression of the envelope protein by the addition of
 tetracycline; 2) positive regulation by removing tetracycline; and,
 optionally, 3) regulated expression of the nucleotide sequence encoding
 the transactivator.
 The packaging cell line of the invention capable of inducible expression of
 a desired envelope protein can be used to produce pseudotyped retroviral
 vectors at pre-centrifugation viral titers of at least more than 10.sup.4
 /ml, preferably more than 10.sup.5 /ml, even more preferably more than
 10.sup.6 /ml. Preferred packaging cell lines of the invention produce
 viral titers of at least 10.sup.5 infectious particles per ml, preferably
 at least 5.times.10.sup.5 per ml, more preferably at least 10.sup.6 per
 ml, and can produce viral titers of 1.times.10.sup.7 per ml or more.
 Preferred packaging cell lines can produce viral titers on the order of at
 least about 1 viral particle/cell, preferably about 10 viral particles per
 cell, more preferably about 100 viral particles per cell or more.
 Preferred packaging cell lines can also be characterized by a viral titer
 produced over a unit of time. For example, preferred packaging cells of
 the invention produce about 10.sup.4 infectious particles per ml per hour,
 preferably about 10.sup.5 particles per ml per hour, and can produce up to
 about 10.sup.6 particles per ml per hr or more.
 The packaging cells can be maintained under conditions that allow for
 production of infectious pseudotyped virions (e.g, in the absence of
 tetracycline) for at least 4 days, preferably at least 7 days, even more
 preferably at least 12 days, and can be maintained for 16 days or longer.
 For example, 293 cells can be maintained at least 7 days after induction
 with high virus productivity (e.g., from about 10.sup.4 to about 10.sup.6
 cfu/ml or greater). Preferably, the packaging cells provide a higher titer
 in a shorter period of viral production time (i.e., as opposed to a lower
 viral titer over a longer period of viral production.
 Preferably, the packing cells of the invention respond within a short time
 after induction, i.e., the packaging cells respond quickly to produce
 pseudotyped virus when tetracycline is removed from the media. Preferably,
 the packaging cells produce detectable viral titers within 48 hours after
 induction, more preferably within 12 hours to 24 hours after induction.
 Preferably, the packaging cells of the invention produce viral titers of
 at least 10.sup.3 cfu/ml, more preferably at least 10.sup.4 cfU/ml, still
 more preferably at least 10.sup.5 cfu/ml, most preferably 10.sup.6 cfu/ml
 or more within 5 days after induction, preferably within 4 days after
 induction, more preferably within 3 days after induction, still more
 preferably within 2 days after induction, and may produce such viral
 titers within 48 hours to 36 hours or less after induction.
 In general, the pseudotyped virus packaging cell lines of the invention can
 produce 10.sup.4 to 10.sup.6 infectious viral particles per ml, provide
 virus production within a short time (e.g., within 24 hours to 48 hours)
 after removal of tetracycline and can produce infectious virus for about 5
 days to about 16 days or more.
 The packaging cells of the invention are preferably derived from cells that
 are easy to maintain in culture, can be grown to high densities, can be
 efficiently transfected using conventional techniques, and can tolerate
 levels of transactivation and desired envelope protein necessary to allow
 inducible expression of viral envelope and production of pseudotyped
 retroviral vectors. Repression of envelope protein production is
 preferably very tight (e.g., even a small amount of tetracycline, results
 in repression of viral envelope expression, and response to induction of
 expression is rapid (e.g., preferably within 12 hours to 36 hours after
 removal of tetracycline). For example, 293 cells grow like regular
 fibroblasts, are easy to culture, and exhibit a high DNA transfection
 efficiency (e.g., by the conventional calcium-phosphate co-precipitation
 method), making genetic manipulation of this cell line very easy.
 Gene Transfer
 In one embodiment, the packaging cells are present within a transgenic
 animal for in vivo production of pseudotyped retroviral particles that can
 be harvested from the animals (e.g., by collecting and isolating
 pseudotyped virions from the animal's blood or any other body fluid). The
 nucleotide sequence(s) encoding the retroviral Gag and Pol proteins and
 the nucleotide sequence(s) encoding the inducible expression system can be
 used to generate transgenic animals according to methods well known in the
 art as described above. The animals can then be infected with infectious,
 replication defective retroviral virions containing the nucleotide
 sequence of interest, which can then infect the "in vivo" packaging cells
 present in the transgenic animal to produce high titers of pseudotyped
 retroviral vector particles. Expression of the envelope protein encoded by
 the inducible expression system can be regulated by administration of
 tetracycline to the transgenic animal as described above.
 In another embodiment, the present invention provides a method for
 introducing an exogenous non-HIV polynucleotide into the chromosome of a
 recipient cell. The method includes contacting a recipient cell with a
 recombinant lentivirus produced by the method of the invention; and
 integrating the exogenous non-HIV polynucleotide into the chromosome. The
 pseudotyped retroviral vector particles generated using the packaging
 cells of the invention can be used to facilitate delivery of a nucleotide
 sequence of interest to a host cell either in vitro or in vivo. For
 example, the pseudotyped retroviral vector particles can be used in gene
 therapy applications to deliver therapeutic gene product-encoding sequence
 to a subject, e.g., a mammalian subject, preferably a human subject. The
 pseudotyped retroviral vector particles can also be used to develop
 various disease or development animal or in vitro models. Recipient cells
 for delivery of lentiviral or retroviral vector particles of the invention
 include endothelial cells, myeloid cells, bone marrow cells, stem cells,
 lymphocytes, hepatocytes, fibroblasts, lung cells, muscle cells, embryonic
 cells, and neuronal cells. Methods for administration of retroviral
 particles to a subject to accomplish in vivo transformation are well known
 in the art (see, e.g., Mulligan 1993 Science 260:926; Anderson 1992
 Science 256:808; Miller 1992 Nature 357:455; Crystal 1995 Science
 270:404). Methods for transformation in vitro using retroviral particles
 are also well known in the art.
 A variety of genes or DNA fragments can be incorporated into the retroviral
 vector particles of the invention for use in gene therapy. Protein of use
 in gene therapy include various hormones, growth factors, enzymes,
 lymphokines, cytokines, receptors and the like.
 Among the genes that can be transferred in accordance with the invention
 are those encoding polypeptides that are absent, are produced in
 diminished quantities, or are produced in mutant form in individuals
 suffering from a genetic disease. Other genes of interest include those
 that encode proteins that have been engineered to circumvent a metabolic
 defect or proteins that, when expressed by a cell, can adapt the cell to
 grow under conditions where the unmodified cell would be unable to
 survive, or would become infected by a pathogen.
 In addition to protein-encoding genes, the present invention can be used to
 introduce nucleic acid sequences encoding medically useful RNA molecules
 into cells. Examples of such RNA molecules include anti-sense molecules
 and catalytic molecules, such as ribozymes.
 Retroviral vector particles of the invention can be used for ex vivo gene
 therapy in accordance with various techniques known in the art. In general
 terms, these techniques involve the removal of target cells of interest
 from a patient, incubation of the target cells with the retroviral vector
 particles, and reintroduction of the transduced target cells into the
 patient. Techniques for administration of retroviral vector particles
 produced by the packaging cells of the invention include those described
 in U.S. Pat. No. 5,580,766. This patent and its teachings are herein
 incorporated by reference in its entirety.
 Methods of Detecting and Modulating HIV Integrase Activity
 At present, HIV integrase is the only viral enzyme for which clinically
 useful inhibitors are not available. Thus, the development of an assay
 which utilizes preintegration complexes (PICs) represents an important
 tool for identifying integration inhibitors. Typically, screens are
 conducted using purified integrase protein alone, but such assays are only
 partially reflective of integration in vivo. The use of retroviral vectors
 obviates the biohazard associated with HIV-1 PICs and the vector cell line
 is a more robust and convenient source of viral particles. The present
 invention provides a method for screening PICs in a relatively high
 through-put fashion.
 Accordingly, the invention provides a method of detecting a viral nucleic
 acid sequence integrated into a target nucleic acid sequence by reversibly
 immobilizing the target nucleic acid sequence to a solid support,
 contacting the target nucleic acid with a viral preintegration complex
 (PIC) containing the viral nucleic acid sequence, under such conditions
 and for sufficient time so as to allow the viral nucleic acid sequence to
 integrate into the target nucleic acid sequence and detecting the
 integrated viral nucleic acid sequence.
 The invention further provides a method of detecting viral integrase
 activity contained in viral preintegration complex (PIC) by reversibly
 immobilizing a target nucleic acid sequence to a solid support; contacting
 the target nucleic acid with the viral preintegration complex (PIC)
 containing a viral nucleic acid sequence, under such conditions and for
 sufficient time so as to allow the viral nucleic acid sequence to
 integrate into the target nucleic acid sequence and detecting the
 integrated viral nucleic acid sequence. The presence of the viral nucleic
 acid sequence in the target nucleic acid sequence is indicative of
 integrase activity.
 In one embodiment, the invention provides a method of identifying a
 compound that affects viral integrase activity contained in a
 preintegration complex (PIC). The method includes reversibly immobilizing
 a target nucleic acid sequence to a solid support; contacting the target
 nucleic acid with the viral preintegration complex (PIC) containing a
 viral nucleic acid sequence and a test compound suspected of affecting
 integrase activity, under such conditions and for sufficient time so as to
 allow the viral nucleic acid sequence to integrate into the target nucleic
 acid sequence; and detecting the integrated viral nucleic acid sequence.
 The amount of viral nucleic acid sequence integrated in the target nucleic
 acid sequence is indicative of the effect of the compound on integrase
 activity.
 Thus, the invention provides a method for identifying a compound which
 modulates integrase activity. The method includes incubating components
 comprising the compound, integrase, a nucleic acid to be integrated and a
 target nucleic acid under conditions sufficient to allow the components to
 interact and determining the effect of the compound on the activity of the
 integrase before and after incubating in the presence of the compound.
 Preferably, the integrase is HIV integrase. HIV integrase can be isolated
 as a part of a preintegration complex (PIC). Compounds that modulate HIV
 integrase activity can include peptides, peptidomimetics, polypeptides,
 chemical compounds and biologic compounds. The term "modulate", as used
 herein, encompasses any means by which integrase activity can be affected,
 such as inhibition of activity or an increase in activity. The activity of
 the integrase can be assayed using methodology as described in Example 3
 (e.g., based on PCR and agarose gel electrophoresis) or by other standard
 methods known in the art.
 Incubating includes conditions which allow contact between the test
 compound, the integrase and the target nucleic acid. The test compound may
 optionally be a combinatorial library for screening a plurality of
 compounds. Compounds identified in the method of the invention can be
 further evaluated, detected, cloned, sequenced, and the like, either in
 solution or after binding to a solid support, by any method usually
 applied to the detection of a specific DNA sequence such as PCR, oligomer
 restriction (Saiki, et al., Bio/Technology, 3:1008-1012, 1985),
 allele-specific oligonucleotide (ASO) probe analysis (Conner, et al.,
 Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligation assays
 (OLAs) (Landegren, et al., Science, 241:1077, 1988), and the like.
 Molecular techniques for DNA analysis have been reviewed (Landegren, et
 al., Science, 242:229-237, 1988).
 The method of the invention includes combinatorial chemistry methods for
 identifying chemical compounds that bind to integrase or affect integrase
 expression or activity. By providing for the production of large amounts
 of HIV integrase, one can identify ligands or substrates that bind to,
 modulate, affect the expression of, or mimic the action of HIV integrase.
 Areas of investigation are the development of therapeutic treatments. The
 screening assay described herein identifies compounds that provide
 modulation of HIV integrase function in targeted organisms. Of particular
 interest are screening assays for compounds that have a low toxicity for
 humans. A wide variety of assays may be used for this purpose, including
 labeled in vitro protein-protein binding assays, protein-DNA binding
 assays, electrophoretic mobility shift assays, immunoassays for protein
 binding, and the like. The purified protein may also be used for
 determination of three-dimensional crystal structure, which can be used
 for modeling intermolecular interactions and transcriptional regulation,
 for example.
 The term "compound" as used herein describes any molecule, e.g. protein or
 pharmaceutical, with the capability of altering the physiological function
 or expression of HIV integrase. Generally, a plurality of assay mixtures
 are run in parallel with different compound concentrations to obtain a
 differential response to the various concentrations. Typically, one of
 these concentrations serves as a negative control, i.e. at zero
 concentration or below the level of detection.
 Where the screening assay is a binding assay, one or more of the molecules
 may be joined to a label, where the label can directly or indirectly
 provide a detectable signal. Various labels include radioisotopes,
 fluorescers, chemiluminescers, enzymes, specific binding molecules,
 particles, e.g. magnetic particles, and the like. Specific binding
 molecules include pairs, such as biotin and streptavidin, digoxin and
 antidigoxin. For the specific binding members, the complementary member
 would normally be labeled with a molecule that provides for detection, in
 accordance with known procedures.
 A variety of other reagents may be included in the screening assay. These
 include reagents like salts, neutral proteins, e.g. albumin, detergents,
 etc. that are used to facilitate optimal protein-protein binding and/or
 reduce non-specific or background interactions. Reagents that improve the
 efficiency of the assay, such as protease inhibitors, nuclease inhibitors
 and anti-microbial agents may be used. The mixture of components are added
 in any order that provides for the requisite binding. Incubations are
 performed at any suitable temperature, typically between 4 and 40.degree.
 C. Incubation periods are selected for optimum activity, but may also be
 optimized to facilitate rapid high-throughput screening. Typically between
 0.1 and 1 hours will be sufficient.
 The present invention provides a method useful for detecting viral
 integration by attaching a target nucleic acid to a substrate associated
 with a solid support and contacting the target nucleic acid with PICs. It
 is envisioned that any substrate useful for conjugating a target nucleic
 acid to a solid support can be used in the present method. Such
 conjugation is preferably a chemical conjugation. Chemical conjugation
 useful for attaching a nucleic acid to a particular substrate can be
 accomplished by any method known to those of skill in the art. Such
 methods may include, for example, chemical conjugation of a nucleic acid
 to a substrate by a disulphide bridge. In the preceding example, the
 nucleic acid-substrate complex can be disrupted by the addition of a
 reducing agent such as, for example, beta mercaptoethanol. Another example
 of chemical conjugation can include a double-stranded target nucleic acid
 having a reactive 5' phosphate covalently bound to amine groups attached
 to a solid support. Subsequent treatment with NaOH disrupts the 5'
 phosphate-amine group conjugate thereby disociating the immobilized target
 nucleic acid-5' phosphate moiety from the amine group attached to the
 solid support.
 Immobilization of substrates to the solid support material may occur via a
 variety of methods apparent to the skilled artisan. Solid supports useful
 for the present method are known to those of average skill in the art. The
 term "solid support", as used herein, is any structure which provides a
 supporting surface for the attachment of a substrate. Well known solid
 supports useful for the method of the invention include, but are not
 restricted to, glass beads, silica aerogels, agarose, Sepharose, Sephadex,
 nitrocellulose, polyethylene, dextran, nylon, natural and modified
 cellulose, polyacrylamide, polystyrene, polypropylene, and microporous
 polyvinylidene difluoride membrane. It is understood that any material
 which allows for the attachment and support of a target nucleic acid is
 included in the present invention. In Example 3 provided herein, a
 96-multiwell plate was utilized as the solid support.
 The following examples are intended to illustrate but not limit the
 invention. While they are typical of those that might be used, other
 procedures known to those skilled in the art may alternatively be used.
 EXAMPLES
 The following examples are put forth so as to provide those of ordinary
 skill in the art with a complete disclosure and description of how to
 carry out the invention and is not intended to limit the scope of what the
 inventors regard as their invention. Efforts have been made to ensure
 accuracy with respect to numbers used (e.g., amounts, temperatures, etc.),
 but some experimental error and deviation should be accounted for. Unless
 indicated otherwise, parts are parts by weight, molecular weight is weight
 average molecular weight, temperature is in degrees Centigrade, and
 pressure is at or near atmospheric.
 EXAMPLE 1
 MATERIALS AND METHODS
 Plasmid Construction
 pSKVG was constructed by cloning the EcoRI VSV-G (Indiana serotype) from
 pMDG( Naldini et al., (1996) Science 272, 263-267) into the EcoRI site of
 Bluescript SK+ (Stratagene, La Jolla, Calif.). The GFP coding fragment was
 excised from pEGFP-N1 (Clontech) by SacI/NotI digestion and ligated to
 SacI/NotI fragment of Bluescript SK+ to create pSKGFP. A PstI fragment
 containing the GFP coding region from pSKGFP and NheI/EcoRV fragment
 containing the VSV-G coding region from pSKVG were ligated to PstI and
 XbaI/PvuII site in pBI (Clontech 6152-1) respectively, to create pBIGFVG.
 BamHI/BglII fragment containing a minimal CMV immediate-early gene promoter
 linked to seven tandem copies of the tetR-binding site replaced the CMV
 promoter(BglII/BamHI fragment), in pcDNAneo to create phCMVn.
 pPTK was constructed by ligation of BglI/SacII fragment encoding all the
 HIV-1 proteins from pDR8.2 (Naldini et al., supra) to BglI(partial)/SacII
 fragment from phCMVn. The XhoI fragment containing the neomycin resistance
 gene was deleted from ptTet-Off (Clontech K1620-A) to create ptTADn from
 which the a fusion protein containing the carboxy terminus of the
 tetracycline repressor and the herpes simplex virus VP16 transactivation
 domain is expressed under the control of the CMV promoter.
 Generation of Lentiviral Vector Packaging Cell Line
 All the cell lines in this study were maintained in Dulbeco's modified
 Eagle's containing tetracycline-free 10% fetal calf serum (Clontech
 8630-1). To generate the stable cell line SODk0 that expresses the fusion
 protein tetracycline repressor /VP16 transactivation domain, human 293
 embryonic kidney cells were cotransfected with 20 mg of ptTADn and 1 mg of
 pSRaBSR that expresses the blastocydine resistance gene, by a calcium
 phosphate precipitation method (Naldini et al., supra). Individual cell
 colonies were selected under 20 mg/ml of blsatocydine. The colonies were
 screened for tTA expression by transient transfection with 20 mg of
 pBIGFVG. Isolation of the 4 highest producer colonies was based on the
 number of fused cells and the level of GFP expression as determined by
 fluorescence microscopy. In order to determine which of the 4 colonies is
 most suitable for vector production the colonies were transfected with 5
 mg of pBIGFVG, 10 mg pPTK, and 15 mg of HrcmvGFP (Miyoshi, et al. (1997)
 Proc. Natl. Acad. Sci. USA 94, 10319-10323.) 48 hours post transfection
 condition media was collected and viral titers on 293 cells were
 determined by serial dilution. The colony which was found to be the
 highest producer in 2 experiments (titer on 293&gt;2.times.10.sup.6 I.U/ml)
 was chosen for generating the stable lentiviral vector packaging cell
 line.
 The lentiviral vector stable packaging cell line SODk1 was generated by
 transfection of SODk0 cells with 10 mg of pPTK, and 10 mg of pBIGFVG. The
 transfected cells were selected for neomycin resistance (400 mg/ml) in the
 presence of 0.7 mg/ml doxycycline. Individual colonies were screened for
 HIV-1 p24 and GFP production and cell fusion in the presence or absence of
 doxycycline as follows. Cells from confluent 10 cm plate were split by
 ratio of 1/4 into PolyLysine precoated plates. Induced cells were cultured
 in the absence of doxycycline. Cell media was changed daily. Control cells
 were cultured in the presence of 0.7 mg/ml doxycycline. On day 4 post
 induction the levels of HIV-1 p24 in conditioned media was assayed by
 ELISA (DuPont). Cell fusion as a marker for VSV-G production, and GFP
 production was determined by fluorescence microscopy. The colony which was
 found to be negative for p24, and GFP, production in the presence of
 doxycycline and yet showed the highest levels of p24 production (&gt;800
 mg/ml) with more than 90% green cells upon induction was chosen as the
 packaging cell line for the lentiviral vector.
 The lentiviral vector producer cell line SODk1CGFI was generated by
 transducing SODk1 cells with HRcmvGFP lentiviral vector at M.O.I of 2.
 Vector Production From SODk1CGFI Cell Line
 To test vector production capability, SODk1CGFI cells were split from
 confluent 10 cm plate into precoated Polylysine plate by ratio of 1/4 in
 the absence of doxycycline. 24 hours after split, the cells were washed
 twice with PBS and refed with doxycycline free media that contained 5 mM
 sodium butyrate. Doxycycline free media that contained 5 mM sodium
 butyrate was replenished daily. Induced SODk1CGFI conditioned media was
 filtered through a 0.45-mm-pore size filter and assayed daily for vector
 titers and p24 concentration in by serial dilutions on 293 cells and by
 p24 ELISA respectively.
 In order to further concentrate the vector, conditioned media was collected
 3 days after addition of sodium butyrate filtered as described before, and
 ultracentrifuged at 50,000.times. g for 2 hours. The pellet was
 resuspended and incubated for 2 hours at 37.degree. C. in Tris-buffered
 saline (TBS) containing 10 mM MgCl.sub.2, four dNTPs (0.1 mM each), 3 mM
 spermine, and 0.3 mM spermidine. After a second ultracentrifugation at
 50,000.times. g for 2 hours the was resuspended in TBS with 2 mg/ml
 Polybrene. The concentrated vector was assayed for p24 concentration, and
 titered on 293 cells as described before.
 Western analysis was performed to test inducibility of VSV-G protein and
 HIV-1 Vpr production in SODk1CGFI cells. Induced ( 3 days after adding
 sodium butyrate) and non induced cells (cultured in the presence of
 doxycyline) were lysed and proteins denatured by boiling for 10' in a
 buffer containing 68 mM tris (pH 6.8), 50 mM NaCl, 0.5 mM EDTA, 0.5
 aprotinin, 50 mg/ml PMSF, 1.5% SDS, 5% glycerol, 5% b-mercaptoethanol. 20
 mg of the denatured protein, were separated on 12.5% polyacrylamide gel
 containing SDS and blotted onto Immobilon-P membrane (Millipore), After
 blocking with 5% nonfat milk in PBS, 0.2% Tween20, for 15' the membranes
 were incubated with mouse monoclonal anti VSV-G (Sigma V-5507) or with
 rabbit HIV-1 Vpr (National Institute of Allergy and Infectious Diseases
 AIDS Research and Reference Reagent Program. reagent 3252) and then with
 goat anti-mouse Ig HRP (pierce), or donkey anti-rabbit horseradish
 peroxidase( Amersham) respectively. The protein bands were detected with
 an ECL kit (Amersham).
 Transduction of Non-dividing Cells in Vitro
 Serial dilution of induced SODkCGFVGI conditioned media were used to
 transduce HeLa cells arrested by culturing in the presence 15 mg/ml
 aphidicolin for 12 hours, HeLa cells cultured in the absence of
 aphidicolin, human embryo fibroblasts arrested by culturing for 48 hours
 in 0.1% fetal calf serum, and human embryo fibroblasts cultured in 10%
 fetal calf serum. Titers were scored 48 hours post transduction by number
 of GFP positive foci divided by the dilution factor.
 Transduction of Terminally Differentiated non Dividing Cells in Vivo
 Adult female Fischer 344 rats were anesthetized as described (Naldini et
 al., supra) and 3 ml of concentrated vector (1.times.10.sup.9 I.U/ml) were
 injected into the left striatum (anterior posterior, +0.2; medial lateral,
 -3.5; dorsal ventral, -4.5) with a 5-ml Hamilton syringe. After 4 weeks
 the animals were sacrificed and perfused with 75 ml saline followed with
 200 ml 4% paraformaldehyde, and 0.2% glutaraldehyde. The fixed brains were
 saturated in 30% sucrose, frozen and sectioned into 50-mm slices.
 Primary antibodies were pooled in TBS with 10% donkey serum and 0.3% Triton
 X-100 and incubated for 48 hours at 4.degree. C.
 EXAMPLE 2
 Induction of SODk1 cells by doxycycline withdrawal in the presence of 5 mM
 sodium butyrate resulted in the production of HIV-1 proteins as determined
 by p24 ELISA. HIV-1 p24 could be detected from day 1 post induction to day
 6 post induction after which significant cell death could be observed.
 Maximal levels of p24 were &gt;800 mg/ml was detected at days 2 and 3 post
 induction. No p24 could be detected in non induced cells.
 GFP production could be detected from day 2 post induction. Cell fusion as
 indicator for VSV-G production could be observed from day 3 post
 induction. Neither cell fusion nor GFP production could be detected by
 fluorescence microscopy in non-induced cells.
 SODk1CGFI Cells
 Induction of SODk1CGFI cells as described before resulted in the production
 of HIV-1 proteins as determined by p24 ELISA. HIV-1 p24 could be
 determined from days 1 to day 6 post induction. Maximal p24 levels (&gt;1000
 mg/ml) could be detected on day 2-3 post induction. Production of HIV-1
 Vpr in induced cells was determined by western analysis.
 VSV-G protein production in induced cells was determined by western
 analysis. Vector production by induced cells could be detected from day 1
 to day 6 post induction. Maximal titer of 1.5.times.10.sup.6 I.U as
 determined by serial dilution on 293 cells could be obtained on days 2-3
 post infection. The vector could be concentrated by ultra centrifugation
 to 1.times.10.sup.9 I.U.
 The vector was shown to be able to transduce non-dividing cells in vitro
 and in vivo. Vector titers on cultured arrested cells was not different
 from the titers on dividing cells. Injection of the vector into adult rat
 brain resulted in the transduction of terminally differentiated
 non-dividing neurons (for methods, see Cohen et al, 1996, Science 272:195,
 incorporated by reference in its entirety).
 Production of HIV-1 p24and Vpr proteins could not be detected in
 non-induced cells by ELISA or western analysis respectively.
 Production of VSV-G protein could not be detected by western analysis in
 non-induced cells. Vector production could not be detected in non-induced
 cells.
 EXAMPLE 3
 The invention further involves using an HIV-based vector produced by a cell
 line as described in the preceding Examples, to establish assays for
 retroviral integration in vitro. Supernatants containing HIV- based vector
 particles were derived from packaging cell lines as described herein.
 Exemplary packaging cell lines are derived from 293 HeLa, Cf2Th, D17,
 MDCK, or BHK cells, most preferably from 293 cells. The viral particles
 from these cell lines were concentrated and used to infect 293T target
 cells. The 293T cells were subsequently lysed and viral replication
 intermediates recovered. (See for example, (Farnet and Haseltine, PNAS
 87:4164, 1990; Ellison et al., J. Virol., 64:2711, 1990, incorporated by
 reference). These intermediates, designated "preintegration complexes"
 (PICs) contain viral cDNA bound to viral and cellular proteins. One such
 viral protein, integrase, directs the covalent connection of viral cDNA to
 target DNA. Accordingly, the present method utilizes PICs to effect in
 vitro covalent integration of viral cDNA into an added target nucleic
 acid. Such methods are useful in the pharmaceutical industry for the rapid
 screening of integrase inhibitors and are of particular interest in HIV
 research.
 The integration assay described here utilized 96-well microtiter plates. As
 shown in FIG. 1, linear, double-stranded target DNA having a reactive 5'
 phosphate (indicated by a dot) was covalently attached to an amine-coated
 microtiter well (FIG. 1A). The amine groups are represented by "N".
 HIV-based preintegration complexes (PICs) were added to the wells (FIG.
 1B). The integration reaction yielded the covalent attachment of the viral
 cDNA (represented by the thick lines) 3' ends to target DNA. Unintegrated
 HIV cDNA molecules were washed away. The DNA strands were then released by
 NaOH denaturation (FIG. 1D). Integrated HIV nucleic acid contained in the
 released sequences were detected and quantified (FIGS. 1E and FIG. 2).
 Integration products released from amine plates were amplified by PCR
 using primers complementary to HIV sequences and detected using agarose
 gel electrophoresis (FIG. 2). The results of five integration reactions
 are shown in FIG. 2: standard conditions, lane 1; with added 20 mM EDTA,
 lane 2; 15 mM MgCl2, lane 3; 0.1% SDS, lane 4; and 0.5% Tx-100, lane 5.
 Standard conditions were 150 mM KCl, 20 mM Hepes pH 7.4, 5 mM MgCl2, 5%
 DMSO, and 0.2% BSA. A DNA marker, lane M, and copy number standards of HIV
 sequences are shown to the right.
 Although the invention has been described with reference to the presently
 preferred embodiment, it should be understood that various modifications
 can be made without departing from the spirit of the invention.
 Accordingly, the invention is limited only by the following claims.