The dimer initiation sequence stem-loop of human immunodeficiency virus type 1 is dispensable for viral replication in peripheral blood mononuclear cells

Abstract

Human immunodeficiency virus type 1 (HIV-1) contains two copies of genomic RNA that are noncovalently linked via a palindrome sequence within the dimer initiation site (DIS) stem-loop. In contrast to the current paradigm that the DIS stem or stem-loop is critical for HIV-1 infectivity, which arose from studies using T-cell lines, we demonstrate here that HIV-1 mutants with deletions in the DIS stem-loop are replication competent in peripheral blood mononuclear cells (PBMCs). The DIS mutants contained either the wild-type (5'GCGCGC3') or an arbitrary (5'ACGCGT3') palindrome sequence in place of the 39-nucleotide DIS stem-loop (NL(CGCGCG) and NL(ACGCGT)). These DIS mutants were replication defective in SupT1 cells, concurring with the current model in which DIS mutants are replication defective in T-cell lines. All of the HIV-1 DIS mutants were replication competent in PBMCs over a 40-day infection period and had retained their respective DIS mutations at 40 days postinfection. Although the stability of the virion RNA dimer was not affected by our DIS mutations, the RNA dimers exhibited a diffuse migration profile when compared to the wild type. No defect in protein processing of the Gag and GagProPol precursor proteins was found in the DIS mutants. Our data provide direct evidence that the DIS stem-loop is dispensable for viral replication in PBMCs and that the requirement of the DIS stem-loop in HIV-1 replication is cell type dependent.

FIG. 1.

Schematic representation of the DIS stem-loop sequences in the HIV-1 RNA genome. The numerical values are based on the RNA nucleotide position of NL4.3. The deletion sequences are indicated as dotted lines. The italic font highlights the DIS sequences or the palindrome sequences within the stem-loop. This figure is modified from a figure in the study by Berkhout (1).

FIG. 2.

Replication kinetics of wt HIV-1 and DIS stem-loop mutants in PHA-stimulated PBMCs (A) and T-cell line Sup T1 (B). PHA-stimulated freshly isolated PBMCs or SupT1 cells were infected with either NL4.3 wt (▪) or mutant (NL_GCGCGC [Δ] and NL_ACGCGT [□]) viruses. Supernatants were collected 3, 7, 10, and 14 days after infection, and RT activity in each sample was measured. Results represent means and standard deviations of triplicate samples and are representative of eight and six sets of experiments for PBMCs and Sup T1, respectively.

FIG. 3.

(A) Mutations in DIS stem-loop inhibit virion packaging of genomic RNA. Virion particles were produced by transfecting the indicated proviral DNA into 293T cells. Virion RNA samples that were normalized via a quantitative p24 assay were separated by electrophoresis on a denatured agarose gel. A dilution series of wt virion RNA was used to construct a standard curve. The impact of mutations on virion RNA packaging was visualized by Northern analysis and quantified by phosphorimaging. Results are representative of three sets of experiments. (B) The conformation but not the stability of HIV-1 virion RNA dimers from transfected 293T cells was affected by mutations of the DIS stem-loop. Virion RNA dimers were isolated from transfected 293T cells and prepared as described in Materials and Methods. Viral monomeric and dimeric RNA species were separated on a 1% native agarose gel via electrophoresis after the samples were heated for 10 min at various temperatures (4, 25, 37, 42, 48, and 52°C, lanes 1 to 6, respectively). Viral RNAs were visualized by Northern analysis using a radioactive riboprobe that specifically recognizes HIV-1 RNA. Results are representative of five sets of experiments.

FIG. 4.

The conformation but not the stability of HIV-1 virion RNA dimers isolated from PBMCs was affected by mutations of the DIS stem-loop. Virion RNA dimers were isolated from infected PBMCs and prepared as described in Materials and Methods. Viral monomeric and dimeric RNA species were separated on a 1% native agarose gel by electrophoresis after the samples had been heated for 10 min at various temperatures (4, 25, 37, 42, 48, and 52°C, lanes 1 to 6, respectively). Viral RNAs were visualized by Northern analysis with a radioactive riboprobe that specifically recognizes HIV-1 RNA. Results are representative of three sets of experiments.

FIG. 5.

Mutations in the HIV-1 DIS stem-loop do not alter the intracellular viral protein and virion protein profile. Intracellular (293T cells) and virion proteins were resolved by SDS-10% PAGE. The viral proteins were visualized by Western blotting using pooled HIV-1-positive patient sera and anti-human horseradish peroxidase-conjugated secondary antibody. Results are representative of five sets of experiments.

FIG. 6.

Replication kinetics of wt HIV-1 and DIS stem-loop mutants passaged through PBMCs in Sup T1 cells. SupT1 cells were infected with either NL4.3 wt (▪) or mutant viruses (NL_GCGCGC [Δ] and NL_ACGCGT [□]) that had been passaged through PBMCs. Supernatants were collected 3, 7, 10, and 14 days after infection, and RT activity in each sample was measured. Results represent means and standard deviations of triplicate samples and are representative of eight sets of experiments.