We have identified proteins that specifically bind the effector domain of Rev while it is bound to the Rev response element (RRE). These proteins are being excised from SDS gels for sequencing and cloning. Using similar, but much smaller scale binding technologies, we have developed an in vitro binding assay based on the cooperative interaction of the HIV-1 Rev protein, its target sequence, the RRE and previously unidentified cellular factors. Using this assay we have detected in crude extracts a nuclear activity that cooperatively binds with the Rev protein to the RRE. This activity should be crucial for the in vivo function of Rev, the emergence from microbiological latency, and the synthesis of mature virions during acute infection. Chromatographic analysis of the activity has determined it consists of at least four and possibly more than five separate proteins. One of the activities has been purified to the extent of five prominent bands on an SDS gel. We should soon be able to sequence and clone it. We have acquired sufficient physicochemical data about the other factors to facilitate their rapid cloning. We are now studying the factors by microinjecting them into mammalian cells along with Rev-dependent indicator genes. In related work we have studied the interaction of the Rev protein with the RRE in the absence of other proteins. We have found that RRE elements determine the binding of Rev molecules subsequent to the first binding event and that Rev must dimerize before binding the RRE. Along slightly different lines, we have used subtractive cloning techniques to isolate clones of a number of cellular genes specifically up- and down-regulated by HIV infection. Surprisingly, HIV infection turns on Line-1, an endogenous human retrovirus-like element, and switches off expression of S16 ribosomal protein. As an extension of our previous work on cell-type-specific viral tropism and the RRE, we have identified other viral determinants of tropism, particularly vpu and vif. During the course of these studies we have discovered a novel interaction between the vpu and vif genes.
