Sequences Downstream of the 5′ Splice Donor Site Are Required for both Packaging and Dimerization of Human Immunodeficiency Virus Type 1 RNA

Abstract

Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5 splice donor site at nucleotide (nt) 289 have been shown to act as packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a "kissing-loop" mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5 splice donor site, are required for efficient RNA packaging and dimerization.Human immunodeficiency virus type 1 (HIV-1) contains a diploid RNA genome that is noncovalently associated in dimer form at its 5Ј ends in a parallel orientation. These attached viral RNA regions were first described as the dimer linkage structure in monkey sarcoma virus (18), and this term has been used subsequently to describe dimerization in other retroviruses. Two models have been proposed to illustrate molecular interactions that constitute the HIV-1 dimer linkage structure. The first involves a tetra-stranded RNA structure, termed a G-tetrad, that is formed by G-rich RNA sequences (23). This structure has been implicated in maintaining the integrity of chromosome telomeres in which stretches of G-rich nucleotide sequences are present (12). G-rich RNA regions were also identified at the 5Ј end of HIV-1 RNA downstream of the major splice donor site. It was therefore hypothesized that formation of G-tetrad structures may contribute to the maintenance of RNA dimers; this notion has been supported by studies performed with synthetic viral RNA fragments, yet has not been extensively tested in the context of the full-length viral RNA genome (2,13,23,42).The second model involves a "kissing-loop" mechanism and is derived from the observation that the stem-loop 1 (SL1) RNA segment, located upstream of the 5Ј spl...

Structural Determinants and Mechanism of HIV-1 Genome Packaging

“…The Lever group first predicted that the CU rich region forms a long-range interaction with the AG rich linker between the Ψ and AUG hairpins 72 , and supporting evidence has more recently been obtained by mutagenesis 235 and SHAPE probing 104,233 , Figure 5. Kjems and coworkers predicted that the CU rich region forms a small hairpin with the nucleotides right after the PBS2 stem loop 9 .…”

“…The ability to bind specifically and tightly to exposed guanosine bases was proposed to be a primary function of the HIV-1 NC zinc knuckles 132,133 . Surprisingly, although mutations that disrupt the stem structure of the Ψ hairpin severely impair genome packaging, substitution of the NC binding GGAG loop by GCUA or AAGA did not significantly affect packaging or replication 235 . Both substitutions contain a tetraloop guanosine, and oligoribonucleotide Ψ RNAs containing these mutations are capable of binding NC (albeit with weaker affinities of ~ 4 μM and ~ 800 nM, respectively) (Heng and Summers, unpublished results).…”

“…Hayashi et al reported that a fragment of the 5′-UTR containing the Ψ hairpin is capable of directing the packaging of heterologous RNAs into virus-like particles 69 , but this finding has not been independently verified. Russell and co-workers reported that the Ψ hairpin and downstream GA rich region are required not only for packaging but also for genome dimerization 235 . Deletion of Ψ, and mutations designed to disrupt base pairing in the stem, have been reported to lead to significant reductions in genome packaging 47,49,59,235 , whereas compensatory mutations designed to restore base pairing in the stem 47 , and the complete substitution of Ψ by a different NC-binding RNA fragment 254 , largely restored packaging efficiency.…”

Global Changes in the RNA Binding Specificity of HIV-1 Gag Regulate Virion Genesis

“…4C, D, S4A). However, there was a tendency for read densities to increase towards the 3’ end of the viral genome, perhaps reflecting the incorporation of spliced viral RNAs into virions generated by Ψ-deleted genomes (Clever and Parslow, 1997; Houzet et al, 2007; Russell et al, 2003). Importantly, these results indicate that multiple sites on the viral genome bind to Gag independently of Ψ and facilitate HIV-1 genome packaging.…”

Cell biology of retroviral RNA packaging

“…[8][9][10]. For HIV-1 and MLV the major packaging determinants are located downstream of the 5' splice donor site that generates all the viral spliced mRNAs 21 ( Fig. 1).…”