Total cellular RNA was isolated from HIV-1-infected and uninfected macrophage by the RNAzol method. Polyadenylated mRNA was purified by centrifugation of the total RNA through oligo(dT) cellulose spin columns. These mRNAs were used to make directional cDNA libraries in lambda Uni-Zap XR. Phagmid libraries were produced by in vivo excision, biotinylated and used for subtractive hybridization. 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). Clones that strongly hybridize to the amplified subtracted probe will be tested for differential expression by Northern blot hybridization using cDNA probes produced from mRNAs from infected and uninfected cells. Promising clones 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 or down-regulated (subtraction will be done in both directions) upon viral infection should provide useful insight into the mechanism of viral replication and pathogenesis.
