Many retroviruses undergo genetic alterations in vivo which may affect the viral oncogenicity, cellular tropism, or immune elimination. For example the transforming avian and murine retroviruses have acquired genetic sequences of the host which are directly oncogenic. Murine leukemia viruses (MuLVs) readily undergo recombination with mouse genomic sequences to generate variants which exhibit ah altered host range of infectivity and can activate cellular oncogenic genes. Point mutation of lentiviruses such as the equine infectious anemia virus (EIAV) or the human immunodeficiency virus (HIV) may contribute to the generation of variants which escape the host immune response. The major emphasis of this project is the occurrence and mechanisms of genetic alteration in retroviruses and the consequences of such alterations. In previous studies we have examined the tissue- specific expression of host range variants which are generated after inoculation of an MuLV. Since the generation of such variants results in a mixed infections of viruses with different host ranges, there is a potential for viral pseudotyping to occur in which the genome of one virus is encapsulated in the virion coat of another. We have found a distinct pattern of pseudotyping which correlates with the onset of leukemia in mice infected with the lymphocytic leukemia virus M-MuLV. In both the spleen and thymus, host range variants are completely pseudotyped by the M-MuLV during most of the preleukemic phase of disease. Thus, all virions exhibit the host range of M-MuLV, regardless of which viral genome they contain. Complete pseudotyping persists in the spleen throughout the course of infection. However, in the thymus a high proportion of virions which exhibit the variant host range arise immediately receding the earliest onset of leukemia. Further studies will focus on the relationship of this burst of variant virions to malignant transformation. Studies on the point mutation rate of retroviruses have determined the rate of viable progeny viruses to be approximately 20-fold slower than previous reports. This study is currently being extended to determine the in vivo polymerase error rate for an MuLV. Several proviruses which have undergone a single replication cycle have been molecularly cloned into a vector which allows both direct DNA sequencing as well as in vitro transcription and RNA fingerprinting. Analysis of these clones will yield the first in vivo polymerase error rate determination for a eukaryotic virus. In previous studies numerous monoclonal antibodies (mAb) directed at MuLV antigens have been developed. We have initiated collaborative experiments with Dr. Pincus of the LMSF to construct and test ricin A-chain immunotoxins (IT) for their ability to selectively kill cells infected by MuLVs. Several immunotoxins exhibit cytotoxic activity for infected cells. These include an immunotoxin which exhibits significant cytotoxicity of cells infected with all classes of MuLVs and another immunotoxin which exhibits virtually complete killing of cells infected with a highly neurovirulent MuLV.
