Retroviruses are single-stranded RNA viruses. Upon infection of a cell by a retrovirus the retroviral RNA genome is transcribed into its corresponding double-stranded DNA by a reverse transcriptase enzyme which is coded for by the viral genome. This DNA then enters the nucleus and integrates into the host DNA using an integrase enzyme which is also coded for by the viral genome. The integrated viral DNA ("proviral" DNA) becomes a component of the host genome, replicating with it and being passed on to daughter cells in a stable manner. The proviral DNA is also in general transcribed to give viral RNA molecules which code for the major viral proteins, namely the gag, pol and env proteins (the "packaging proteins"). The full length RNA transcript can be packaged by the viral proteins into a viral particle which then buds off in a piece of cell membrane, in which are embedded env-derived peptides. This membrane-coated viral particle is a fully competent viral particle and can go on to infect other cells.
The genome of a retrovirus (in either the RNA or DNA form) can be divided conceptually into two parts. The first, or "trans-acting," category consists of the regions coding for viral proteins. These include the group specific antigen ("gag") gene for synthesis of the core coat proteins, the "pol" gene for the synthesis of various enzymes (such as reverse transcriptase), and the envelope ("env") gene for the synthesis of envelope glycoproteins. Other proteins may also be produced in different retroviruses from messages produced by various internal splicing reactions. These viral functions determine a considerable part of the host specificity of a virus. In the murine leukemia virus ("MLV") family, for example, the env gene products interact with cell surface receptors and determine whether the virus is ecotropic (infects only mice and rats), xenotropic (infects non-mouse species only), or amphotropic (infects mouse and other species, including human), and it has been suggested that the host range of a virus can be altered by replacing its env protein (see Milewski, Recombinant DNA Technical Bulletin, Volume 9, Number 2, page 88 (1986)). The gag gene products define specificity in mice with respect to two main types: N (NIH derived mice) or B (Balb/C derived mice).
In general, the second part of the retroviral genome is referred to as the "cis-acting" portion and consists of the regions which must be on the genome to allow its packaging and replication. This includes the packaging signal on an RNA molecule, such as the viral RNA, which identifies that RNA molecule to viral proteins as one to be encapsidated, Long Terminal Repeats ("LTRs") with promoters and polyadenylation sites, and two start sites for DNA replication. The promoters, enhancers, and other regions of the LTRs are also capable of conferring tissue specificity such that the virus will only be "expressed" (i.e., transcribed and translated) in specific cell types even though it may infect others.
It has been recognized that the cis-acting elements are grouped at either end of the viral genome, in or near the LTRs. Thus, the internal or "trans-acting" part of a cloned provirus might be replaced by a gene of choice to create a "vector construct" (see for example, F. Ledley, The Journal of Pediatrics, Vol. 110, No. 1, p. 1 (January, 1987)). When the vector construct is placed in a cell where necessary viral proteins are present, transcribed RNA should be packaged as viral particles which, in turn, will bud off from the cell. These particles will be indistinguishable in appearance from native virus particles, although they carry only the RNA of the vector construct into a cell and integrate it within that cell's genome. It is believed that the gene will then be functional in the new cell but, without the trans-acting part of the viral genome, will be incapable of expressing those proteins required for further virus production. Hence, the vector construct and the virus carrying it are "replication defective" normally being unable to produce new viral particles in the cell. The vector construct can, however, be transcribed and express its gene product.
Retroviruses are known to be widely spread in non-human species and the cause of various pathogenic conditions. However, only a very small number of retroviruses have been identified in humans, and these only recently. In addition, new retroviral sequences have been detected in cells of human origin (see Callahan et al., Science, Vol. 228, p. 1208; 1985), although their significance remains unknown.
The cause of many human pathogenic conditions remains unknown. One such pathogenic condition is human rheumatoid arthritis ("RA"). In various studies, viruses have been found to be present in body fluids from some human subjects suffering from RA, who were studied as part of a larger group of RA subjects. However, the results in such studies have been inconclusive in that, typically, a majority of the subjects did not exhibit the presence of the virus identified in the minority. For example, parvovirus has been demonstrated in RA synovial fluid in a minority of RA suffering patients. Annals of The Rheumatic Diseases, 46:219-223 (1987). Epstein-Barr ("EB") virus infection also appears to be present in a majority of patients with RA, but in no higher percentage than in the normal population, although RA patients are apparently less able to regulate such infections than normal control subjects (Bardwick et al., Arthritis .Rheum., Vol. 23, p. 626, 1980). A retrovirus has been identified by Brassfield et al., Arthritis Rheum, Vol. 25, p. 930 (1982), as the apparent cause of caprine arthritis, a goat arthritis clinically similar to human rheumatoid arthritis.
Previously, the standard techniques used in an effort to determine the presence of an unknown retrovirus (e.g. one whose presence may be suspected but which has not previously been isolated or characterized) have been a reverse transcriptase assay, electron microscopy, and various immunologic and nucleic acid hybridization assays. For example, such techniques have been described by Weiss, R. (1982) in RNA Tumor Viruses Vol. I, pages 209-260 (Cold Spring Harbor Laboratory, N.Y.).
Complementation assays have been used to titer retroviruses that have already been isolated and characterized. In other words, complementation assays are used to determine the ability of a known retrovirus to have a specific biologic effect. For example, in the S+ L- assay a murine leukemia virus rescues a MSV replication-deficient virus, infects an untransformed cell line and thereby induces a transformation event, Eckner and Kettrick, J. of Virology, Vol. 24, p. 383-90 (1979). Such assays are time consuming, are highly dependent on the ability of the viruses to complement and the ability of the recipient cell to be transformed, and are subjective in view of the necessity to assess cell morphology associated with the transformed state. In addition, these assays deal with known, characterized retroviruses and are used simply to titer those viruses.
As pointed out by Norval et al., Ann. Rheum. Dis., Vol. 38, p.507 (1979) and Hart et al., Ann. Rheum. Dis., Vol. 38, p. 514 (1979), the presence of a retrovirus in a majority of patients with RA has not been demonstrated, despite extensive efforts to determine the cause of RA. The failure to detect a retrovirus which is clearly associated with RA, or retroviruses associated with other human pathogenic conditions of unknown etiology, could be the result of relative insensitivity of both the reverse transcriptase assay and electron microscopy, and the limitation of immunologic and nucleic acid hybridization assays which only search for virus proteins or nucleic acid sequences very closely related to those known to exist. Further, many retroviruses can only grow in a few specific cell types, further exacerbating the difficulty in detecting previously unknown retroviruses. For example, it is known that human T-cell lymphotrophic viruses types I and II (HTLV I and HTLV II) can only be effectively grown in certain cells such as in T-cell growth factor-driven lymphocytes, or cell lines derived therefrom, as described by Broder et al., Ann, Rev. Imunol., Vol. 3, p. 321 (1985).
Retroviral constructs (i.e. retroviruses carrying vector constructs) such as those described by Gruber et al., Science, Vol. 230, p. 1057-1061 (1985), including pLPLM, have been developed as vehicles for gene replacement therapy. The plasmid pLPLM, a murine leukemia virus-derived construct, has retroviral promoters (LTRs) and packaging signals, but the pol, gag and env genes have been deleted and replaced by cDNA for human hypoxanthine phosphoribosyltransferase ("HPRT"). A. D. Miller et al., Proc. Natl. Acad. Sci. USA, ("PNAS") Vol. 80, p. 4709-4713 (1983); J. K. Yee, Gene, Vol. 53, p. 97-104. It is known that when viral constructs having the LTRs, packaging signal, and a gene of interest, are placed in a cell having a genome coding for a retrovirus with the packaging signal deleted (known as a "helper cell"), such retrovirus will pseudotype the RNA transcribed from the vector construct, which carries a packaging signal, into a recombinant retrovirus (a retrovirus carrying a replication defective retroviral vector, is sometimes referred to herein as a retroviral particle). See, e.g., PCT application No. WO 86/00922, published Feb. 13, 1986; Molecular Biology of Tumor Viruses, Second Edition, "RNA Tumor Viruses" Robert Weiss (Ed) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA (1984); Mann et al., Cell, Vol. 33, p. 153-159 (May 1983); Watanabe et al., Molecular and Cellular Biology, Vol. 3, No. 12, p. 2241-2249 (Dec., 1983); Watanabe et al., Proc. Natl. Acad. Sci., USA, Vol. 79, p. 5986-5990 (October, 1982); and Watanabe et al., Eukaryotic Viral Vectors, p. 115-121, editor Y. Gluzman, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA (1982). In addition, replication defective retroviral genes have been rescued from cells by infecting the cells with a suitable retrovirus known to be present. Even if the defective vector sequences are not appropriate to the rescuing viral proteins, such rescue happens at an appreciable frequency compared to the frequency of a fully compatible defective vector (0.01%-1%). For example, see Linial, Journal of Virology, Vol. 38, No. 1, p. 380-382 (April, 1981). Since a fully compatible defective vector will give signals of about 10.sup.6 units/ml, frequencies described above should be easily detectable.