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 Watson-Crick 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.

FIG. 1.

Schematic representation of the viruses used in this study and the mutations created within SL1 of the 5′ untranslated region of HIV-1. (A) General structures of the nearly full-length HIV-1 vectors that make up the recombination system. (B) Proposed structure of the HIV-1 SL1. (C) Extension of the KL complex down the stems of SL1 results in a more stable ED conformation. (D) Design of the DIS mutants containing altered sequences in the loop of SL1 and their impact on the base pairing within the KL complex of each virus combination. (E) SL1 SM and the double mutants (SM3A, SM3B) containing both the stem and DIS mutations. (F) Base pairing of the SL1 SM virus with another SL1 SM virus or with the wild-type (WT) virus in either the wild-type DIS or mutant context. Black letters, HIV-1 sequences; red letters, mutated bases. LTR, long terminal repeat.

FIG. 2.

Virus production, RNA packaging efficiency, and virus infectivity of DIS mutants. Virus was produced by transient transfection of 293T cells with wild-type plasmid, pHIVBHO, or the DIS mutant plasmids and pIIINL(AD8)env, encoding HIV-1 Env. (A) Viral particle production as measured by p24 levels. Virus was harvested from the producer cells 48 h posttransfection and assayed for p24 levels by enzyme-linked immunosorbent assay. (B) Efficiency of viral RNA encapsidation. Viral RNA was extracted from a sample of each virus, reverse transcribed, analyzed by real-time PCR using a gag-specific primer-probe set, and normalized to p24 levels. (C) Virion infectivity. Titers of virus were determined on TZM reporter cells containing a Tat-responsive luciferase gene and normalized to p24 levels. All three graphs are plotted as a percentage of the wild-type (WT) levels; shown are the means ± standard errors of results from three independent transfection experiments. Also shown are the DIS sequences and the number of complementary bases within the putative KL complex for each virus assayed.

FIG. 3.

Recombination frequency of SL1 mutants. (A) The role of the DIS sequence in RNA partner selection. The recombination rates of the DIS mutant viruses with viruses containing the identical DIS sequence compared with that of two viruses harboring complementary DIS mutations. “A or B” signifies that results were obtained from two cell lines, one containing two A mutant viruses and the other containing two B mutant viruses. For example, “1A or 1B” represents results compiled from a cell line containing two 1A viruses and a cell line containing two 1B viruses. “A + B” signifies that results were obtained from two cell lines both containing an A mutant virus and a B mutant virus. For example, “1A + 1B” represents results from cell lines containing one 1A and one 1B virus. (B) The impact of dimer extension on partner selection. The recombination rate of the SL1 mutant alone or in the presence of wild-type (WT) virus was measured. Also, the double mutants containing a G- or C-loop DIS and the SL1 SM were assayed for recombination with their equivalent DIS mutant with wild-type SL1 stems. Results are depicted as the means ± standard errors (n = 7) for two independent cell lines each transfected and assayed at least three times; P-values, generated by Student's t test, indicate significant deviation from the wild-type recombination rate. The recombination rate of wild-type virus (gray columns) previously measured using the same recombination system is shown for comparison.

FIG. 4.

Effect of SL1 mutations on template-switching frequency. (A) Strategy to measure template switching within the IRES region. The presence of two markers, hsa or thy and gfp, within the viral genomes provides an internal control for the rate of template switching during reverse transcription. After reconstitution of gfp, the RT complex will synthesize the IRES region and then the thy gene. However, an HSA⁺ GFP⁺ virus will result if an additional template-switching event occurs within the IRES. (B) Relative levels of HSA⁺ and Thy⁺ cells within the GFP⁺ population of infected cells. LTR, long terminal repeat; WT, wild type.

FIG. 5.

Dimerization state of the SL1 mutant viral genomes. (A) Genomic RNA extracted from virus released from a selection of dual infected cell lines. The RNA was phenol-chloroform extracted from harvested virus and separated by nondenaturing gel electrophoresis. Genomic RNA was identified using a ³²P-radiolabeled RNA riboprobe targeting the gag region of HIV-1. Wild-type RNA heat denatured at 85°C was also included as a control. (B) Viral RNA isolated from virions treated with 1 μM lopinavir to block virus maturation. The viral RNA from PR⁻ virus, incapable of maturing its RNA, was also included as a control. WT, wild type.