FUNCTIONS OF LEUKEMIA VIRUS R REGIONS

We have obtained preliminary data that an element exists within the 5' 32 nucleotides of the R components of the genomes of certain murine leukemia viruses (MuLVs) that contributes to the transcriptional activity of the virus. The R regions of the MuLVs, which are situated downstream of the initiation site of the transcription, bear striking similarity to those of the gibbon ape leukemia virus, simian sarcoma virus, and feline leukemia viruses. Our preliminary data indicate that secondary structure of the R sequences is important for the activity of the element. Also, the R region element acts at least in part to increase RNA polymerase II loading onto viral LTR templates, as detected in nuclear run-on assays. We propose here to perform a detailed series of studies to investigate how the MuLV element functions. In particular, we hypothesize that the MuLV R region element functions in a manner analogous to that of the HIV-1 TAR element. Specifically, we hypothesize that a cellular factor exists that is functionally equivalent to HIV-1 Tat and recognizes the MuLV R element at the level of RNA to stimulate transcriptional initiation and/or RNA polymerase processivity. We propose to use a mutagenesis approach to identify precisely which nucleotides within the MuLV R element are critical for activity. A series of experiments will be performed to test whether the MuLV sequences function as a DNA or RNA moiety. We will also determine whether they strictly affect transcriptional initiation or whether they affect processivity of RNA polymerase molecules loaded onto viral transcriptional templates. other viruses will be examined to see whether they contain a similar element and what the sequence context requirements for the function of the MuLV element are. We will also test whether the MuLV sequence can at least partially substitute for the HIV-1 TAR element. Protein-nucleic acid binding assays will be used to look for cellular factors that recognize the MuLV R region element.