The general goal of this research is to understand the role of nucleolar protein B23 as a possible receptor and/or transporter of the HIV-1 Rev protein. Protein B23 (Mr 38,000), is a RNA-associated nonribosomal phosphoprotein and putative ribosome assembly factor. Protein B23 forms a specific complex with the HIV-1 Rev protein and it is suggested that B23 is the nucleolar receptor and/or transporter for Rev. The Rev protein regulates the splicing of viral mRNA so that HIV-1 structural proteins are produced late in the infection. We have characterized two forms of B23 (B23.1 and B23.2) and we are able to produce these by expression in bacteria. We now propose to further characterize the Rev-B23 interactions and to investigate the possible role of B23 in the transport of the Rev protein.
The specific aims are: (a) to determine how Rev binding is affected by B23 isoform structure and posttranslational modification. Relative affinities of natural and bacterially produced B23.1 and B23.2 for Rev will be determined. Similar comparisons will be done with B23.1 phosphorylated in vitro with casein kinase II and with a CDC2 type protein kinase. The effects of B23 isoforms on Rev fiber formation will be investigated by electron microscopy. (b) to determine what part of the B23 sequence interacts with the Rev protein. These experiments will involve competition with synthetic peptides, partial proteolysis and deletion mutations of B23 expressed in bacteria. (c) to test the hypothesis that B23 transports Rev from the cytoplasm to the nucleus or nucleolus. For this we will use a digitonin-permeabilized cell system designed for nuclear import studies. The accumulation of fluorescently labeled Rev and B23 in the nucleus will be measured by fluorescence microscopy and image analyses. The effect of added B23 on the kinetics of Rev accumulation will be measured. Studies will also be performed on covalently cross-linked RevB23 complex to determine whether B23 actually carries Rev into the nucleus or nucleolus. These studies should not only answer questions about the Rev-B23 interactions but also aid in the development of a means of targeting a specific HIV protein by drugs which might interfere with viral replication.
