FIG. 4.

Defective Gag expression from an env mutant provirus is complemented by an infectious MMTV provirus or Rem cDNA. (A) Complementation assay for gag-pol mRNA export. Rectangles represent trim cassettes, and circles represent Rem protein. Lines, some with V shapes to show spliced regions, represent capped and polyadenylated viral RNAs. For simplicity, the doubly spliced rem RNA is not shown in the nucleus. (B) The Gag production defect of the env148 mutant is complemented by cotransfection of an infectious wild-type MMTV provirus. XC cells stably transfected with penv148 (148) or pHYB-MTV (HM) were transiently transfected with pHYB-MTV or penv148, respectively. After fractionation, cytoplasmic and nuclear RNAs were used in RT-PCRs to detect trim-containing unspliced viral RNA (gag-pol-trim), completely spliced sag RNA, or gapd RNA. Negative controls included reactions lacking RNA (lane 1) or RNA from untransfected XC cells (lanes 3 and 8). The penv148 vector (p148) was used as a size marker for the product of unspliced RNA (lane 2). Asterisks represent lanes where complementation occurred. (C) Complementation of the env148 mutant defect by cotransfection with RemGFP or RemΔCGFP expression plasmids. HC11 cells were transiently transfected for 24 h and then incubated with 10⁻⁶ M dexamethasone for an additional 48 h to induce the LTR promoter. Whole-cell lysates were subjected to Western blotting and incubation with Gag-specific (upper panel) or actin-specific antibodies (lower panel).