Abstract
Human immunodeficiency virus type 1 (HIV-1) mRNAs encoding structural proteins contain multiple inhibitory/instability elements (INS), which decrease the efficiency of viral protein expression. We have previously identified a strong INS element (INS-1) within the p17(gag) coding region. Here we show that poly(A)-binding protein 1 (PABP1) binds preferentially to INS-1 within the p17(gag) mRNA, but not to a mutated mRNA in which INS-1 function is eliminated. Competition experiments performed in the presence of different nucleic acids and homoribopolymers demonstrated preferential binding of PABP1 to the INS-1-containing RNA. In contrast to HeLa cells and several lymphoid cell lines, certain human glioma cell lines exhibit high levels of gag expression in the absence of Rev upon transient transfection with wild type gag expression vectors. We analyzed extracts of different cell lines and found that the binding of PABP1 to INS-1 RNA is significantly diminished in glial cell extracts. The expression levels of gag correlate with the absence of binding of PABP1 to the INS-1 RNA in cellular extracts. These results suggest a role for PABP1 in the inhibition of gag expression mediated through INS-1.
Highlights
We further characterize the mechanism of function of the inhibitory sequences
Detection of Specific RNA-Protein Complexes—We prepared in vitro transcribed p17gag RNA containing the region of instability elements (INS)-1 (KS17 RNA), and a mutant RNA transcribed from pKS17M1234 [11], which has inactivated INS-1 (KS17M RNA)
Analysis of RNA-protein complexes obtained from nuclear extracts did not reveal any differences between KS17 RNA or KS17M RNA, but these complexes were distinct from those obtained with RNA-binding region (RRE) RNA (Fig. 1B, lane 3)
Summary
We further characterize the mechanism of function of the inhibitory sequences. We studied the cellular factors interacting with INS-1 using UV-cross-linking of in vitro transcribed RNA to cellular extracts. The ability of PABP1 to bind p17gag RNA in vitro appears to be cell type-specific and correlates with inhibitory effects of INS-1 in vivo. We propose that binding of PABP1 to INS-1 within the gag mRNAs may contribute to the mechanisms regulating HIV-1 expression. Rev functions by facilitating the transport, stability, and translation of partially spliced and unspliced HIV-1 mRNAs that contain a Rev-specific RNA-binding region (RRE) [1,2,3,4,5]. Rev-dependent mRNAs are defective in expression, due to specific RNA regions scattered throughout the gag, pol, and env regions of HIV-1. These regions were named inhibitory sequences (INS/CRS/IN) and were shown to prevent efficient
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