Figure 3

rtm ORF codes for two 18- and 17-kDa protein isoforms related to envelope precursor and TM proteins. A, Relationships between domains shared by Env precursor, Rev and Rtm proteins. Splicing events within the Env coding region leading to rev and rtm ORFs are shown. Env precursor and Rev derived domains are represented by open and shaded boxes, respectively. B, Schematic representation of Rev and Rtm expression constructs. Plasmids pKcRev and pKcRtm are predicted to express singly-spliced mRNAs encoding the Rev and Rtm proteins, respectively. The pKRtm expression vector contains the rtm cDNA generated by RT-PCR from cells transfected with pKcRtm. The approximate positions of PCR primers are indicated (horizontal arrows). C, Coding capacity of the rtm ORF. Transfected 293T cells were radiolabeled 5 h with [³⁵S]-methionine 48 h after transfection, and protein extracts were subjected to immunoprecipitation analysis using rabbit affinity-purified antibodies raised against either the first 38 amino acids of Env precursor (anti-NH₂ Env), the 110-amino acid cytoplasmic domain of TM (anti-CD™), or the 98-amino acid carboxy terminus of Rev (anti-Rev). Immunoprecipitated proteins were resolved by electrophoresis through a SDS-15% polyacrylamide gel and visualized by autoradiography. D, Analysis of in vitro translation products of rtm cDNA. [³⁵S]-methionine labeled polypeptides were synthesized in an in vitro coupled transcription-translation reaction with pGEM-1 (lanes 1 and 2) or rtm cDNA (lanes 3 and 4). Crude products (lanes 1 and 3) and proteins immunoprecipitated with affinity-purified anti-CD™ antibodies (lanes 2 and 4) were analyzed as described above.