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Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity - PubMed

  • ️Fri Jan 01 2016

Stability of HIV Frameshift Site RNA Correlates with Frameshift Efficiency and Decreased Virus Infectivity

Pablo Garcia-Miranda et al. J Virol. 2016.

Abstract

Human immunodeficiency virus (HIV) replication is strongly dependent upon a programmed ribosomal frameshift. Here we investigate the relationships between the thermodynamic stability of the HIV type 1 (HIV-1) RNA frameshift site stem-loop, frameshift efficiency, and infectivity, using pseudotyped HIV-1 and HEK293T cells. The data reveal a strong correlation between frameshift efficiency and local, but not overall, RNA thermodynamic stability. Mutations that modestly increase the local stability of the frameshift site RNA stem-loop structure increase frameshift efficiency 2-fold to 3-fold in cells. Thus, frameshift efficiency is determined by the strength of the thermodynamic barrier encountered by the ribosome. These data agree with previous in vitro measurements, suggesting that there are no virus- or host-specific factors that modulate frameshifting. The data also indicate that there are no sequence-specific requirements for the frameshift site stem-loop. A linear correlation between Gag-polymerase (Gag-Pol) levels in cells and levels in virions supports the idea of a stochastic virion assembly mechanism. We further demonstrate that the surrounding genomic RNA secondary structure influences frameshift efficiency and that a mutation that commonly arises in response to protease inhibitor therapy creates a functional but inefficient secondary slippery site. Finally, HIV-1 mutants with enhanced frameshift efficiencies are significantly less infectious, suggesting that compounds that increase frameshift efficiency by as little as 2-fold may be effective at suppressing HIV-1 replication.

Importance: HIV, like many retroviruses, utilizes a -1 programmed ribosomal frameshift to generate viral enzymes in the form of a Gag-Pol polyprotein precursor. Thus, frameshifting is essential for viral replication. Here, we utilized a panel of mutant HIV strains to demonstrate that in cells, frameshifting efficiency is correlated with the stability of the local thermodynamic barrier to ribosomal translocation. Increasing the stability of the frameshift site RNA increases the frameshift efficiency 2-fold to 3-fold. Mutant viruses with increased frameshift efficiencies have significantly reduced infectivity. These data suggest that this effect might be exploited in the development of novel antiviral strategies.

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Figures

FIG 1
FIG 1

Representation of the wild-type HIV-1 frameshift site and the RNA constructs designed to discern the relative contributions of local stability (ΔGLocal), defined as the predicted free energy of the first 3 bp (including the adjacent stacking interaction with the nearest neighboring pair), and overall RNA stem-loop stability (ΔG) to HIV-1 frameshift efficiency.

FIG 2
FIG 2

Effects of frameshift site mutations on Gag-Pol and Gag levels in cells and in virus-like particles (VLPs). (A and C) HEK293T cells were transfected with plasmids encoding the indicated mutations and cultured in the presence of saquinavir. Cell lysates (A) and virus-like particles (C) were harvested and measured using Western blots probed with a monoclonal anti-p24 antibody as described in Materials and Methods. (B and D) The ratio of Gag-Pol to Gag was used to calculate frameshift efficiencies in cells (B) and to quantify the Gag-Pol/Gag ratio in VLPs (D). *, P < 0.001; #, P < 0.05 (compared with WT values). Horizontal dashed lines represent the median value for the WT. (E) Overall thermodynamic stability versus frameshift efficiency. Data were fitted to a one-phase exponential decay function, Y = 0.045 × e−0.2X + 6.7. (F) Local stability of the first 3 bp versus the frameshift. Data were fitted to a one-phase exponential decay function, Y = 0.004 × e−0.8X + 7.8. (G) Correlation between Gag-Pol and Gag levels observed in cell lysates and purified VLPs.

FIG 3
FIG 3

Effects of mutations in the anchoring helix on HIV-1 frameshifting and Gag/Gag-Pol levels in VLPs. (A) The wild-type HIV-1 secondary structure and mutations in M9 (bold) that disrupt the anchoring helix. (B to E) The ratio of Gag-Pol/Gag was determined as described in the Fig. 2 legend. #, P < 0.05 (compared with WT values).

FIG 4
FIG 4

Analysis of a putative secondary slippery site. (A) Secondary structures of RNAs and mutations (bold). -SS contains a double U-to-C mutation to knock out the primary slippery site. -SS2 also includes additional double U-to-C mutations to disrupt the putative secondary slippery site. M10 is a single C1680U mutation, while M11 has a knocked-out primary slippery site and a C1680U mutation. (B to E) Determination of frameshift efficiencies for these mutants performed as described for Fig. 2A. *, P < 0.001; #, P < 0.05 (compared with WT values).

FIG 5
FIG 5

(A) Effects of frameshifting mutants on Gag/Gag-Pol processing. (B and C) Gag processing was evaluated by Western blot analysis for cell lysates (B) and purified VLPs (C). Normalization was carried out using the total amount of p55, p41, and p24 for each mutant relative to the WT. Data shown represent averages of results from 4 individual experiments. *, P < 0.001; #, P < 0.05 (compared with WT values).

FIG 6
FIG 6

Viral infectivity as measured by a single-cycle infectivity assay. (A) Representative images of the infectivity assay for the WT and each mutant. (B) Viral GFP expression was quantified and normalized relative to the WT virus. Two different negative controls were used: WT-VSV (cells transfected only with the WT vector and without VSV-G protein expression vector) and neg (no transfected cells). Four single-cycle infectivity assays were carried out. *, P < 0.001; #, P < 0.05 (compared with WT values).

FIG 7
FIG 7

Immunofluorescence of capsid (p24) and matrix (p17) expression in HEK 293T cells transfected with the WT and frameshift mutants M1 to M6. Cells were fixed 48 h posttransfection. Differences between WT and the frameshift mutants in capsid (p24) expression do not appear to have affected Gag localization within the cell. Viral GFP expression data indicate transfected cells.

FIG 8
FIG 8

Quantification of the expression of reverse transcriptase (RT). (A and C) Representative blots probed with the polyclonal anti-RT antibody and the monoclonal anti-p24 antibody using cell lysates (A) or purified VLPs (C). (B and D) Relative expression levels of RT in cell lysates (B) and in purified virus-like particles (D) were calculated using the total amount of p55, p41, and p24 for each construct and were normalized to the WT. *, P < 0.001; #, P < 0.05 (compared with WT values).

FIG 9
FIG 9

Electron micrographs of immature and mature HIV-1 particles from WT cells and M1, M2, M4, M5, and M6 mutant cells.

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