NMR Studies of the Structure and Function of the HIV-1 5′-Leader

Abstract

The 5′-leader of the human immunodeficiency virus type 1 (HIV-1) genome plays several critical roles during viral replication, including differentially establishing mRNA versus genomic RNA (gRNA) fates. As observed for proteins, the function of the RNA is tightly regulated by its structure, and a common paradigm has been that genome function is temporally modulated by structural changes in the 5′-leader. Over the past 30 years, combinations of nucleotide reactivity mapping experiments with biochemistry, mutagenesis, and phylogenetic studies have provided clues regarding the secondary structures of stretches of residues within the leader that adopt functionally discrete domains. More recently, nuclear magnetic resonance (NMR) spectroscopy approaches have been developed that enable direct detection of intra- and inter-molecular interactions within the intact leader, providing detailed insights into the structural determinants and mechanisms that regulate HIV-1 genome packaging and function.

Increasing the CpG dinucleotide abundance in the HIV-1 genomic RNA inhibits viral replication

“…The core packaging signal for HIV-1 is in the 5′ UTR; however, approximately 300 nt of the 5′ end of gag has been proposed to improve viral titre [7,8]. We did not codon modify the first 21 nt of gag because this region is under purifying selection [11,12] and NMR structures have shown that it base pairs with the U5 region of the 5′ UTR to form the dimer promoting conformation of the gRNA that is packaged into virions [10,76]. However, the relative importance of the sequence in gag beyond the first 21 nt for packaging the full-length HIV-1 gRNA, as opposed to heterologous transcripts, is unclear.…”

“…The gRNA can be divided into three regions: a 336 nt 5′ untranslated region (UTR), a 219 nt 3′ UTR, and an 8618 nt region that is densely packed with multiple open reading frames (nt lengths reference the HIV-1 NL4-3 strain [9]). The 5′ UTR contains several cis-acting elements in complex stem-loop structures that regulate multiple stages of the viral life cycle including transcription, splicing, gRNA dimerization, encapsidation and reverse transcription [8,10]. The central 8618 nt region encodes nine open reading frames: gag, pol, vif, vpr, tat, rev, vpu, env and nef.…”

HIV-1 initiates genomic RNA packaging in a unique subset of host RNA granules

“…Where indicated lysates were analyzed by velocity sedimentation and or IP, as described above except that IPs for gRNA quantification were washed four times in detergent buffer and once in non-detergent buffer. For total cell lysate analysis, aliquots corresponding to ∼5 x10 3 COS-1 cells, 2 x10 4 293T cells and 8.5 x10 3 HeLa-MCP-GFP cells were used. For VLP analysis, aliquots corresponding to VLPs from 1 x 10 5 293T cells and 1.5 x10 4 HeLa-MCP-GFP cells were used.…”

“…For total cell lysate analysis, aliquots corresponding to ∼5 x10 3 COS-1 cells, 2 x10 4 293T cells and 8.5 x10 3 HeLa-MCP-GFP cells were used. For VLP analysis, aliquots corresponding to VLPs from 1 x 10 5 293T cells and 1.5 x10 4 HeLa-MCP-GFP cells were used. For gradient analysis, ∼4 x10 5 COS-1 cells, 2 x 10 6 203T cells, or 6 x 10 5 H9 cells were analyzed on a single 5 ml gradient.…”

“…In addition to being used for packaging, HIV-1 gRNA in the cytoplasm is also used for translation of Gag and GagPol (reviewed in [1]). Translation and packaging are thought to be mutually exclusive, with the shift between these processes likely controlled by a gRNA conformational switch (reviewed in [3,4]). Thus, gRNAs that are packaged are likely to be nontranslating gRNAs with unique structural features [5].…”