Importance of ribosomal frameshifting for human immunodeficiency virus type 1 particle assembly and replication - PubMed (original) (raw)
Importance of ribosomal frameshifting for human immunodeficiency virus type 1 particle assembly and replication
M Hung et al. J Virol. 1998 Jun.
Abstract
The recent development and use of protease inhibitors have demonstrated the essential role that combination therapy will play in the treatment of individuals infected with the human immunodeficiency virus type 1 (HIV-1). Past clinical experience suggests that due to the appearance of resistant HIV-1 variants, additional therapeutics will be required in the future. To identify new options for combination therapy, it is of paramount importance to pursue novel targets for drug development. Ribosomal frameshifting is one potential target that has not been fully explored. Data presented here demonstrate that small molecules can stimulate frameshifting, leading to an imbalance in the ratio of Gag to Gag-Pol and inhibiting HIV-1 replication at what appears to be the point of viral particle assembly. Thus, we propose that frameshifting represents a new target for the identification of novel anti-HIV-1 therapeutics.
Figures
FIG. 1
HIV-1 gene expression and frameshift signal. (A) The full-length HIV-1 mRNA encodes two precursor polyproteins, Gag and Pol, that are cleaved into their individual proteins during assembly of the mature viral particle. Abbreviations: MA, matrix; CA, capsid; NC, nucleocapsid; PR, protease; RT, reverse transcriptase; IN, integrase. (B) The HIV-1 frameshift signal is composed of two parts, the slippery sequence (UUUUUUA) and a stem-loop, a region of stable secondary structure in the mRNA. ORF, open reading frame.
FIG. 2
In vitro HIV-1 frameshifting assay. (A) Schematic representation of the RNA constructs used in the in vitro frameshifting assay. In the FS construct, functional luciferase is produced only when frameshifting occurs at the HIV-1 frameshift signal. The L17 construct contains a modified HIV-1 frameshift signal so when translated it produces the same protein as does the FS construct but without the requirement for frameshifting. (B) Chemical structure of RG501, which was provided by D. Boykin (Georgia State University). (C) RG501 stimulates frameshifting in vitro. Translations of either FS (▪) or L17 (□) RNA were performed in the presence of a range of RG501 concentrations (all at a final DMSO concentration of 0.8%). The amount of luciferase generated from each translation, as determined by light output measured in RLUs, was normalized to that from translations performed in the presence of DMSO alone.
FIG. 3
RG501 affects frameshifting at multiple viral frameshift signals. (A) Proposed frameshifting signals from other retroviruses. The ribosomal frameshift signals are shown for HIV-2 gag-pol, SIV gag-pol, HTLV-I GP, and HTLV-I PP. (B) RG501 stimulates frameshifting at multiple frameshift signals. Translations were performed in the presence of RG501 as described in Materials and Methods. For each pair of RNAs representing the different viral frameshift signals, at a given RG501 concentration, the percent frameshifting was determined by comparing the light output derived from the FS RNA with the light output derived from the L17 RNA. The percent frameshifting for each RG501 concentration was then compared to the percent frameshifting for the same viral constructs in the absence of RG501 and expressed as a fold increase in frameshifting HIV-1 (◊), HIV-2 (□), SIV (⧫), HTLV-I GP (•), and HTLV-I PP (▪).
FIG. 4
Cell-based HIV-1 frameshifting assay. (A) Schematic representation of the RNA constructs used in the cell-based frameshifting assay. The constructs were designed along similar lines as the in vitro constructs. In the FSP construct, functional SEAP (4) is produced only when frameshifting occurs. The S17 construct contains a modified HIV-1 frameshift signal so that when translated it produces the same protein as the FSP construct but without the requirement for frameshifting. (B) RG501 stimulates frameshifting in cells. COS cells were transfected with either FSP (▪) or S17 (□) expression vectors. After incubation in the presence of RG501 (final DMSO concentration, 0.5%) for 48 h, SEAP levels were determined with a chemiluminescence SEAP kit (Tropix). The RLUs for each concentration of RG501 were normalized to those from cells incubated in medium supplemented with 0.5% DMSO.
FIG. 5
RG501 inhibits acute HIV-1 replication. CCRF-CEM cells were infected (MOI = 0.1) with either HIV-1IIIB (○) or HIV-1RTMDR (□) for 1 h. Infected and uninfected cells were incubated in the presence of a range of concentrations of RG501 for 7 to 10 days. The levels of infectious virus (○ and □) were determined with a p24 ELISA and the cellular toxicity profile of RG501 (▪) was determined with a tetrazolium assay.
FIG. 6
Stimulation of frameshifting is detrimental to HIV-1 replication. (A) Increased frameshifting inhibits viral production from chronically infected cells. After CH-1 cells were incubated in the presence of RG501 for 72 h, the levels of viral particles were determined with a standard p24 ELISA (Cellular Products). In these experiments, neither RG501 nor AZT had any discernible cellular toxicity and each data point represents the average of at least four samples. (B) Immunoblot analysis of Gag p24 immunoreactive products present in viral pellets from chronically infected cells. Proteins (5 μl of viral pellet) were separated by electrophoresis in 4 to 12% gradient polyacrylamide–sodium dodecyl sulfate (SDS) gels and immunoblotted with a Gag p24 polyclonal antibody. Lanes: 1, untreated COS cells; 2, untreated CH-1 cells; 3, RG501 (500 μg/ml)-treated CH-1 cells. (C) Immunoblot analysis of human HIV-1 immunoglobulin-reactive products present in CH-1 cell lysates. After incubation in the presence of RG501, CH-1 cell lysates were prepared by using a Triton buffer. CH-1 cell lysates (5 μg of protein) from either untreated (lane 1) or RG501 (500 μg/ml)-treated (lane 2) cells were separated by electrophoresis in 4 to 12% gradient polyacrylamide–SDS gels and immunoblotted with human HIV-1 immunoglobulin. (D) Immunoblot analysis of RT immunoreactive products present in cell lysates. Lysates (5 μg of protein) prepared as for panel C were separated in 6% polyacrylamide–SDS gels and immunoblotted with a polyclonal antibody raised against RT. Samples represent lysates of untreated (lane 1) or RG501 (500 μg/ml)-treated (lane 2) CH-1 cells and of untreated (lane 3) or RG501 (500 μg/ml)-treated (lane 4) 12A2 cells. The numbers to the left of panels B and D represent the relative positions of molecular size markers (in kilodaltons), and the numbers to the right of panels B to D indicate the positions of viral proteins p55 (Gag), p160 (Gag-Pol), p50s (MA, CA, and NC with or without p1 spacer), p41 (MA, CA), and p24 (CA).
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