Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro.

Abstract

The diploid genome of all retroviruses is made of two homologous copies of RNA intimately associated near their 5' end, in a region called the dimer linkage structure. Dimerizatlon of genomic RNA is thought to be important for crucial functions of the retroviral life cycle (reverse transcription, translation, encapsidation). Previous in vitro studies mapped the dimer linkage structure of human immunodeficiency virus type 1 (HIV-1) in a region downstream of the splice donor site, containing conserved purine tracts that were postulated to mediate dimerization, through purine quartets. However, we recently showed that dimerization of HIV-1 RNA also involves sequences upstream of the splice donor site. Here, we used chemical modification interference to identify nucleotides that are required in unmodified form for dimerization of a RNA fragment containing nucleotides A ubiquitous property of retroviruses is that their genonme consists of two homologous copies of single-stranded RNA (1). Electron microscopy showed that these two RNAs are joined together in an apparent parallel orientation by a structure called the dimer linkage structure (DLS) located near their 5' end (2-5). Dimerization of genomic RNA is considered to control several essential steps of the retroviral life cycle. First, it was proposed to act as a positive signal for encapsidation (6,7). Second, it was suggested to downregulate the translation ofthe gag gene in Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) (8, 9). Third, the dimeric nature of the retroviral genome is thought to be of importance in the process of reverse transcription and recombination since it may account for the first strand transfer and template switching during proviral DNA synthesis (10-14). Therefore, dimerization of genomic RNA most likely represents a potential target for the design of antiviral drugs against HIV and other retroviruses.However, the process of dimerization is still poorly understood. Dimerization of synthetic fragments containing the (29). Again, probing data indicate that the same structure is found in synthetic RNA fragments (9,30) and in genomic RNA extracted from infected cells (29).In HIV-1, several reports showed that a RNA region of -100 nucleotides located downstream of the splice donor (SD) site is able to dimerize in vitro (6,(21)(22)(23)(24). This region that includes important components of the packaging signal (29,31,32) was assumed to contain the putative DLS. However, the mechanism of dimerization is still a subject of controversy. In an initial study, we proposed that polypurine tracts, the only common motifs found in the putative dimerization-encapsidation region of most retroviral RNAs, may be involved in the dimerization process through the formation of quartets involving both guanines and adenines (21). This concept has been disputed by recent reports suggesting that dimers formed with short RNA restricted to the postulated DLS are stabilized by quartets containing only guanines (23,24). However, the di...

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA

“…Thus, in MMTV, a combination of both cis -acting sequences and structural elements are responsible for the recognition and differential packaging of the unspliced RNA during the encapsidation process. The case of human immunodeficiency virus type 1 (HIV-1) may somewhat be similar to MMTV: the 5ʹ UTR in HIV-1 implicated in RNA packaging folds into several stem loops, SL1-4 [39,40] of which SL1 harbors a GC-rich 6-nt pal sequence in the form of a loop that has been shown to function as the DIS [41,42]. Until recently SL3, which is present downstream of mSD, had been proposed to be the main HIV-1 packaging signal since it harbors a conserved region containing a GGAG tetraloop that has been shown to bind NC [43].…”

In vitro selection of RNAs that bind to the human immunodeficiency virus type-1 gag polyprotein

“…Different structures and sequences within ψ may be required for distinct dimer formation, gag binding and RNA packaging steps. Guanine quartets (33) and kissing loops (34)(35)(36)(37)(38)(39) have been proposed to be important for some of these steps. We considered whether the nucleocapsid-binding RNAs described here could form such structures.…”

First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions

“…Beside these intramolecular interactions, symmetric intermolecular interactions take place in vitro at the DIS (12)(13)(14) and could also occur at TAR (15) and poly(A) hairpin (16). In addition, tRNA 3 Lys , the reverse transcription primer, is selectively encapsidated into virions and is annealed to the PBS during or after budding (17,18).…”