Dimer Initiation Signal of Human Immunodeficiency Virus Type 1: Its Role in Partner Selection during RNA Copackaging and Its Effects on Recombination

Abstract

Frequent human immunodeficiency virus type 1 (HIV-1) recombination occurs during DNA synthesis when portions of the two copackaged RNAs are used as templates to generate a hybrid DNA copy. Therefore, the frequency of copackaging of genomic RNAs from two different viruses (heterozygous virion formation) affects the generation of genotypically different recombinants. We hypothesized that the selection of copackaged RNA partners is largely determined by Watson-Crick pairing at the dimer initiation signal (DIS), a 6-nucleotide palindromic sequence at the terminal loop of stem-loop 1 (SL1). To test our hypothesis, we examined whether heterozygous virion formation could be encouraged by manipulation of the DIS. Three pairs of viruses were generated with compensatory DIS mutations, designed so that perfect DIS base pairing could only occur between RNAs derived from different viruses, not between RNAs from the same virus. We observed that vector pairs with compensatory DIS mutations had an almost twofold increase in recombination rates compared with wild-type viruses. These data suggest that heterozygous virion formation was enhanced in viruses with compensatory DIS mutations (from 50% to more than 90% in some viral pairings). The role of the SL1 stem in heterozygous virion formation was also tested; our results indicated that the intermolecular base pairing of the stem sequences does not affect RNA partner selection. In summary, our results demonstrate that the WatsonCrick pairing of the DIS is a major determinant in the selection of the copackaged RNA partner, and altering the base pairing of the DIS can change the proportion of heterozygous viruses in a viral population. These results also strongly support the hypothesis that HIV-1 RNA dimers are formed prior to encapsidation.

High efficiency of HIV-1 genomic RNA packaging and heterozygote formation revealed by single virion analysis

“…Here, we provide direct physical evidence that 1 particle contains RNA derived from 2 HIV-1 genomes and that genetically distinct RNAs segregate according to a random distribution. Our study also demonstrates that changing the DIS sequences affects the proportion of the heterozygous virions, which is in general agreement with our genetic recombination studies (9,10). Compared with 2 subtype B viruses with GCGCGC in their DIS sequences, when 1 subtype B virus had GCGCGC in its DIS and 1 virus had GTGCAC in its DIS, we observed decrease in both recombination rate (4-fold) and proportion of heterozygous virions (2.3-fold).…”

“…Based on previous recombination studies, we hypothesized that the mismatch between the DIS sequences from subtype B and subtype C viruses decreased the base-pairing of these 2 RNAs and reduced the formation of heterozygous particles (9). RNA containing GGGGGG and RNA containing CCCCCC in the DIS were inferred to form heterodimers more efficiently than homodimers, thereby increasing the proportion of heterozygous virions in the viral population (10). Using the aforementioned RNA-labeling system, we examined whether similar alterations to the DIS sequences could also change the copackaging of HIV-1 genomes and affect the proportion of heterozygous virions in the viral population.…”

“…In 1 study, monomeric RNAs were detected in samples isolated from newly harvested viruses, leading to the hypothesis that 2 monomeric RNAs were packaged (8). Based on genetic studies, which showed that recombination rates can be regulated by the identity of the dimerization initiation signal (DIS) sequence, we hypothesized that RNA dimerization occurs before encapsidation (9,10). The DIS is a 6-nt palindromic sequence located at the 5Ј untranslated region (UTR) of HIV-1 RNA.…”

“…In contrast, the recombination rates increased significantly when 1 HIV-1 variant contained GGGGGG and the other contained CCCCCC in their DIS. We hypothesized that RNAs from these 2 viruses preferred to form heterodimers, thereby elevating the frequencies of heterozygous virions and the recombination rate (10). Additionally, how RNA genomes are distributed in virions generated from dually infected cells is another biologically important question that has not been directly answered.…”

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

“…Furthermore, it is the presence of these structural motifs that explains the phenomena of co- and cross-packaging among retroviruses that have no relationship to each other [11]. For example, it has been long observed that specificity of packaging can be exchanged in many retroviruses by the substitution of packaging signals that have no sequence homology [16–22]. Therefore, retroviral gRNA packaging process must involve recognition of packaging sequences at the secondary/tertiary structure level(s) rather than only at the sequence level.…”