Total cellular RNA was isolated from HIV-I-infected and uninfected H9 cells by the RNAzol method. Polyadenylated mRNA was purified by centrifugation of the total RNA through oligo(dT) cellulose spin columns. These mRNAs are being used to make directional cDNA libraries in lambda gt22A. Additionally, cDNA libraries will be made in lambda Uni-Zap XR and phagmid libraries will be produced by in vivo excision. Differentially-expressed clones will be isolated from the Uni-Zap libraries, as described by Schweinfest et al. (Gene Anal Tech Appl 1990;7:64-70). The lambda gt22A libraries will be screened using a subtracted probe enriched for infection-specific sequences produced by enzymatic amplification of subtracted cDNAs, as described by Hla and Maciag (Biochem Biophys Res Comm 1990;167:637-43). Clones that strongly hybridize to the amplified subtracted probe will be tested for differential expression by Southern blot hybridization using cDNA probes produced from mRNAs from infected and uninfected cells. Differential expression will be confirmed by RNA gel (Northern) blot assays using filters containing RNA from both infected and uninfected cells and radiolabeled probes from clones that appear promising on the Southern blot assay. Confirmed infection-specific genes will be sequenced and the sequences will be compared to DNA databases in order to determine if they correspond to known genes. Genes that are induced upon viral infection should provide useful insight into the mechanism of viral replication and pathogenesis. Furthermore, they may be useful as diagnostic reagents, targets for immunotherapy, or targets for intracellular vaccination.
