Viral SELEX reveals individual and cooperative roles of the C-box and G-box in HIV-2 replication

Abstract

The 59 UTR of HIV-2 genomic RNA contains signaling motifs that regulate specific steps of the replication cycle. Two motifs of interest are the C-box and the G-box. The C-box is found in the 59 untranslated region upstream of the primer binding site, while the G-box is found downstream from the major splice donor site, encompassing the gag start codon and flanking nucleotides. Together the C-box and the G-box form a long-range base-pairing interaction called the CGI. We and others have previously shown that formation of the CGI affects RNA dimerization in vitro and the positions of the C-box and the G-box are suggestive of potential roles of the CGI in other steps of HIV-2 replication. Therefore, we attempted to elucidate the role of the CGI using a viral SELEX approach. We constructed proviral DNA libraries containing randomized regions of the C-box or G-box paired with wild-type or mutant base-pairing partners. These proviral DNA libraries were transfected into COS-7 cells to produce viral libraries that were then used to infect permissive C8166 cells. The ''winner'' viruses were sequenced and further characterized. Our results demonstrate that there is strong selective pressure favoring viruses that can form a branched CGI. In addition, we show that the mutation of the C-box alone can enhance RNA encapsidation, and mutation of the G-box can alter the levels of Gag protein isoforms. These results suggest coordinated regulation of RNA translation, dimerization, and encapsidation during HIV-2 replication.

Regulation of the activity of the promoter of RNA‐induced silencing, C3PO

“…The DNA library for SELEX was a generous gift from Dr. Steven Lodmell (University of Montana, Missoula, MT) (see Ref. ) . The Ambion MEGAshortscript T7 transcription kit was procured from Life Technologies.…”

Human Retrovirus Genomic RNA Packaging

“…For both HIV-1 and HIV-2, the BMH structure, which is composed of several intramolecular interactions, promotes dimerization by altering the gRNA structure such that the palindromic sequence of the hairpin loop is exposed [ 219 , 220 , 221 , 222 , 223 ]. Both the HIV-1 and HIV-2 BMH structures promote gRNA packaging, which implies a linkage in these two processes [ 146 , 220 , 224 ]. Taken together, these observations suggest a mechanism in which changes in RNA structure may serve as a molecular switch between processes associated with gRNA packaging—i.e., dimerization, NC binding, and viral protein translation.…”