Thriving under Stress: Selective Translation of HIV-1 Structural Protein mRNA during Vpr-Mediated Impairment of eIF4E Translation Activity
Figure 8
Cytoplasmic HIV-1 gag mRNA retains interaction with CBP80 in the cytoplasm.
(A) CEMx174 cells infected with HIV-1 or mock-infected were used to harvest total cellular protein (Total) and the nuclear (Nuc) and cytoplasmic (Cyto) compartments. Equivalent aliquots were immunoblotted with the indicated antiserum. (B) Cytoplasmic lysates from (A) were subjected to immunoprecipitation with CBP80 (top) or eIF4E (bottom) antibodies. Immunoblotting of equivalent aliquots of input and the immunoprecipitated material determined that the IP efficiency was similar between the lysates. Specificity of the IP was verified by using IgG isotype control. (C) Immunoprecipitates from (B) were used to harvest RNA, which were subjected to reverse transcription reactions with or without reverse transcriptase (RT + or −), followed by gene-specific PCR. Products were separated by agarose gel electrophoresis. The gag, nef or GAPDH amplification products from control Total RNA preparation (lanes 1, 2) and RNA-immunoprecipitates (lanes 3–15) are indicated. Lane 15 (No DNA) indicates PCR reaction contained water in place of cDNA.