The human immunodeficiency virus type 1 ribosomal frameshifting site is an invariant sequence determinant and an important target for antiviral therapy

Abstract

Human immunodeficiency virus type 1 (HIV-1) utilizes a distinctive form of gene regulation as part of its life cycle, termed programmed -1 ribosomal frameshifting, to produce the required ratio of the Gag and Gag-Pol polyproteins. We carried out a sequence comparison of 1,000 HIV-1 sequences at the slippery site (UUUUUUA) and found that the site is invariant, which is somewhat surprising for a virus known for its variability. This prompted us to prepare a series of mutations to examine their effect upon frameshifting and viral infectivity. Among the series of mutations were changes of the HIV-1 slippery site to those effectively utilized by other viruses, because such mutations would be anticipated to have a relatively mild effect upon frameshifting. The results demonstrate that any change to the slippery site reduced frameshifting levels and also dramatically inhibited infectivity. Because ribosomal frameshifting is essential for HIV-1 replication and it is surprisingly resistant to mutation, modulation of HIV-1 frameshifting efficiency potentially represents an important target for the development of novel antiviral therapeutics.

FIG. 1.

HIV-1 genome and mutations in the slippery site. (A) HIV-1 genome with the nine major ORFs. Ψ indicates sequences important for genomic RNA packaging. The overlapping region of gag and pol genes contains the −1 ribosomal frameshift signal. LTR, long terminal repeat. (B) Nucleotide sequence of wild-type HIV-1 (strain HXB2) frameshift signal with the slippery site, the spacer, and the putative RNA secondary structure. (C) Sequences of mutations in the slippery site used in the frameshift and infectivity assays. Wt, wild type.

FIG. 2.

Mutations in the HIV-1 slippery site effect programmed −1 ribosomal frameshifting. (A) The dual luciferase construct contains wild-type or mutant HIV-1 frameshift signal inserted into the multiple cloning site (MCS), between the Renilla luciferase (rluc) and the firefly luciferase (fluc) genes. It has simian virus 40 promoter (SV40P) and polyadenylation (SV40pA) sequences. (B) The ratios of expression of two luciferase genes were measured for wild type and mutants and compared to the luciferase activity for the p2luci vector to obtain percent frameshifting. In the wild type, the level of frameshifting is approximately 4 to 6%. In the graph, wild-type levels are set at 100% and compared to those of the slippery site mutants. These are average values from three experiments done in duplicate.

FIG. 3.

HIV-1 infectivity affected by changes in the slippery site. (A) Schematic representation of the vector constructs used for the HIV-1 infectivity assay. We used wild-type HIVgpwt or the mutant gag-pol constructs HIVgpS1 to HIVgpS6. Boxes interrupted with a jagged line symbolize partial deletions; ΔΨ signifies a 33-bp deletion in the encapsidation signal downstream of the splice donor site; CMV, immediate-early promoter from human cytomegalovirus; BGHpA, bovine growth hormone polyadenylation signal; VSV-G, coding sequence for the G envelope protein from VSV; SV40pA, simian virus 40 polyadenylation signal; SNV, spleen necrosis virus U3 promoter; gfp, GFP gene; IRES, internal ribosomal entry site from encephalomyocarditis virus 5′ untranslated region; puro, puromycin resistance gene; LTR, long terminal repeat. (B) Results from viral infectivity assays testing the slippery site mutants in comparison to the wild type. Results were scored as GFP-positive and puromycin-resistant colonies of infected target HeLa cells. Results of viral infectivity assays of slippery site mutants are expressed in IU per milliliter of viral supernatant. These values are averages of four independent transfections. Wt, wild type.

FIG. 4.

Correlation of frameshifting levels with Gag and Gag-Pol polyprotein ratios. The ratios of Gag to Gag-Pol proteins in the wild type and the mutants were assayed using a coupled in vitro transcription and translation (TNT) reticulocyte lysate kit. Please note that the figure depicts one experiment representative of four performed. (A) The sodium dodecyl sulfate-polyacrylamide gel shows the wild type and mutants with each lane marked accordingly. The mutants S1 through S6 are defined in Fig. 1. The positions of the 55-kDa Gag and 160-kDa Gag-Pol polyproteins are indicated. (B) Gag and Gag-Pol protein ratios were calculated from the intensities of protein bands on the gel by densitometry and compared to the wild-type ratio, which was set at 100%. Wt, wild type.