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. 2022 Jan 12;96(1):e0134921.
doi: 10.1128/JVI.01349-21. Epub 2021 Oct 13.

Perturbing HIV-1 Ribosomal Frameshifting Frequency Reveals a cis Preference for Gag-Pol Incorporation into Assembling Virions

Bayleigh E Benner  1   2 , James W Bruce  1   3 , Jacob R Kentala  1 , Magdalena Murray  1 , Jordan T Becker  1 , Pablo Garcia-Miranda  1   4 , Paul Ahlquist  1   3 , Samuel E Butcher  4 , Nathan M Sherer  1
Affiliations

Perturbing HIV-1 Ribosomal Frameshifting Frequency Reveals a cis Preference for Gag-Pol Incorporation into Assembling Virions

Bayleigh E Benner et al. J Virol. .

Abstract

HIV-1 virion production is driven by Gag and Gag-Pol (GP) proteins, with Gag forming the bulk of the capsid and driving budding, while GP binds Gag to deliver the essential virion enzymes protease, reverse transcriptase, and integrase. Virion GP levels are traditionally thought to reflect the relative abundances of GP and Gag in cells (∼1:20), dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event occurring in gag-pol mRNAs. Here, we exploited a panel of PRF mutant viruses to show that mechanisms in addition to PRF regulate GP incorporation into virions. First, we show that GP is enriched ∼3-fold in virions relative to cells, with viral infectivity being better maintained at subphysiological levels of GP than when GP levels are too high. Second, we report that GP is more efficiently incorporated into virions when Gag and GP are synthesized in cis (i.e., from the same gag-pol mRNA) than in trans, suggesting that Gag/GP translation and assembly are spatially coupled processes. Third, we show that, surprisingly, virions exhibit a strong upper limit to trans-delivered GP incorporation; an adaptation that appears to allow the virus to temper defects to GP/Gag cleavage that may negatively impact reverse transcription. Taking these results together, we propose a "weighted Goldilocks" scenario for HIV-1 GP incorporation, wherein combined mechanisms of GP enrichment and exclusion buffer virion infectivity over a broad range of local GP concentrations. These results provide new insights into the HIV-1 virion assembly pathway relevant to the anticipated efficacy of PRF-targeted antiviral strategies. IMPORTANCE HIV-1 infectivity requires incorporation of the Gag-Pol (GP) precursor polyprotein into virions during the process of virus particle assembly. Mechanisms dictating GP incorporation into assembling virions are poorly defined, with GP levels in virions traditionally thought to solely reflect relative levels of Gag and GP expressed in cells, dictated by the frequency of a -1 programmed ribosomal frameshifting (PRF) event that occurs in gag-pol mRNAs. Herein, we provide experimental support for a "weighted Goldilocks" scenario for GP incorporation, wherein the virus exploits both random and nonrandom mechanisms to buffer infectivity over a wide range of GP expression levels. These mechanistic data are relevant to ongoing efforts to develop antiviral strategies targeting PRF frequency and/or HIV-1 virion maturation.

Keywords: Gag; Gag-Pol; HIV; PRF; cis-acting RNA element; protease; reverse transcription; ribosomal frameshift; virion; virus assembly.

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Figures

FIG 1
FIG 1
Increasing PRF element stem-loop stability results in an increase in GP production and enriched levels of GP associated with virions. (A) Representation of the wild-type (WT) HIV-1 gag-pol mRNA frameshift (FS) site secondary structure and that of PRF mutants –SS and M1. M1 was engineered to exhibit elevated stem-loop stability, with ΔGLocal being the predicted free energy of the first 3 bp of the WT and M1 stem-loops. (B) WT, M1, and −SS Gag and GP expression and incorporation into virus particles. HEK293T cells generating WT or the indicated PRF element mutant viruses were cultured in the presence of the protease inhibitor saquinavir (10 μM) to prevent Gag and GP processing. Cell lysates and virions were harvested at 48 h. Gag and GP were detected by Western blotting using anti-p24Gag primary antiserum and infrared-labeled secondary antibodies. HSP90 was detected as a loading control. (C) FS frequency was defined as the ratio of cell-associated GP to Gag (orange bars). GP incorporation frequency into virions was defined as the ratio of virion-associated GP to Gag (blue bars). Fold changes in GP/Gag ratios for the indicated conditions are indicated by black lines, with comparisons derived from three independently performed biological replicates. Error bars represent the standard deviations of the means. *, P < 0.05, **, P < 0.001, and ***, P < 0.0001. P values were derived using Student’s two-tailed t test for comparisons as indicated in the figure, except for −SS P values, which were derived from comparisons of FS frequency and GP incorporation levels to the WT values.
FIG 2
FIG 2
Virus complementation reveals a nonlinear relationship between GP expression and viral infectivity at suboptimal levels of GP. (A) Infectivity of virions produced by WT, M1, and −SS reporter viruses. Viral GFP expression was quantified and normalized to that of WT virus (set to 100%). (B) Schematic of viral 2-color complementation assay wherein WT or PRF mutant GFP reporter viruses were coexpressed in cells generating a Rev− HIV-1 RFP reporter virus encoding a WT PRF element. “FSE” refers to the RNA frameshift regulatory element. (C) Summary of infectious yields of PRF mutant viruses produced alone or coproduced with WT RFP reporter virus in HEK239T cells expressed at a 1:1 ratio. Pseudotyped virus particles were assayed by infecting HEK293T cells (an example is shown) prior to measuring per-well fluorescence intensity relative to the WT control. (D) Data from panel C shown in graphical format. Error bars represent standard deviations of the means derived from three independent biological replicates. (E) Predicted cellular GP/Gag ratios plotted against cumulative relative infectivity, with measurements derived from panels A and D. The dashed green line indicates that a GP/Gag ratio of 0.5 yields 90% overall (GFP plus RFP) infectivity for the −SS+WT condition, close to that of WT virus alone. In contrast, the dashed red line indicates a that a GP/Gag ratio of more than 2-fold yields less infectivity (52%) for the M1+WT condition than WT. Comparisons were derived from three independently performed biological replicates. *, P < 0.05; **, P < 0.001; ***, P < 0.0001; n.s., nonsignificant (for comparisons of M1 and −SS to the corresponding WT using Student’s two-tailed t test [A and D]).
FIG 3
FIG 3
Suboptimal activity of GP supplied in trans suggests a cis preference for GP incorporation into virions. (A) Schematic of GPOnly HIV-1 RFP reporter virus and altered slippery sequence that places pol in the 0 reading frame. “FSRE” refers to the RNA frameshift regulatory element. (B) Western blot analysis of Gag and GP expression confirmed that the GPOnly RFP virus generated GP and not Gag, and at higher levels than GP derived from WT transcripts. Total Gag plus GP protein expression levels were similar for WT and GPOnly transcripts. Note that GP expression alone did not produce virus particles. Hairlines indicate sites where bands from a single immunoblot were spliced together to improve data presentation, but with no further modifications. (C) Effects of GP on viral infectivity and Gag processing when coexpressed with WT, −SS, or M1 viruses. HEK293T cells were transfected with plasmids encoding WT, –SS, or M1 GFP reporter viruses (1,500 ng input plasmid) in the presence or absence of increasing amounts of GPOnly RFP reporter virus plasmid (25, 50, 100, 150, 200, and 300 ng). Viral infectivity was determined as for Fig. 2, with values shown normalized to WT virus production, for three independently performed biological replicates. Both GFP (green) and RFP (red) output is shown for each condition, with error bars representing the standard deviations of the means for both GFP and RFP infectivity, normalized to WT. Cell lysates and virions were also probed for p24Gag by Western blotting as described for Fig. 1B. Hairlines indicate sites where bands from a single immunoblot were spliced together to improve data presentation, but with no further modifications. (D) Virion p24Gag levels were quantified from the experiment reported in panel C and plotted against the corresponding total relative infectivity measurements (GFP and RFP) for each coexpression scenario. Pearson correlation coefficients were determined for each condition. (E to G) Plots depicting expected infectivity (dashed blue lines) based on expected per-transcript GP/Gag ratios (derived as shown in Table 1) and the predictive model in Fig. 2E, relative to actual observed infectivity (orange lines; measured in panel C) for WT, −SS, and M1 coexpression with GPOnly, respectively. Error bars represent the standard deviations of the means for the three biological replicates.
FIG 4
FIG 4
Protease inhibitor treatment confirms cis preference and reveals a strong upper limit to trans-mediated delivery of Gag-Pol into virions. (A) HEK293T cells cultured in the presence of 10 μM saquinavir were transfected to express WT, M1, or −SS viruses (1,500 ng plasmid) in the absence or presence of increasing levels of GPOnly virus (25, 100, or 500 ng plasmid). Cell lysates and virions were harvested at 48 h and probed for Gag and GP levels by quantitative Western blotting as for Fig. 1B. All values were normalized to the WT-only condition (black dashed line). The apparent limit of trans GP incorporation is indicated by the red dashed line. Hairlines indicate sites where bands from a single immunoblot were spliced together to improve data presentation, but with no further modifications. (B) Bar graph showing the fold change in GP incorporation efficiencies calculated from GP band intensities for virions divided by GP associated with cells, from three independent biological replicates, showing greater incorporation of GP when made in cis by WT and M1 viruses. Error bars represent the standard deviations of the means. *, P < 0.05; **, P < 0.001; ***, P < 0.0001 (for comparisons to WT using Student’s two-tailed t test).
FIG 5
FIG 5
Excessive cis-mediated GP incorporation into virions causes premature Gag/GP cleavage and loss of Pol subunits RT and IN from virions. (A) Time course analysis of HIV-1 virions produced from HEK293T cells generating WT or M1 GFP reporter virus at 12, 24, and 48 h posttransfection. Gag cleavage patterns over the experimental time course were analyzed by Western blotting, and blots were probed using anti-p24Gag antisera. Comparison of lanes 4 to 5 and 7 to 8 illustrates the effects of the M1 PRF in increasing GP levels in cells and Gag cleavage kinetics in M1 virions relative to WT virions. (B and D) HEK293T cells generating WT, −SS, or M1 reporter virus were cultured in the absence (vehicle control) or presence of saquinavir, with virions harvested at 48 h posttransfection. GP incorporation was measured by Western blot using either anti-reverse transcriptase (RT) primary antisera (B) or anti-integrase (IN) primary antisera (D). (C and E) Relative levels of GP, RT subunits p51 and p66 (C), or IN (E) in virions for −SS and M1 relative to WT vehicle control or saquinavir treatment (n = 3). Error bars represent the standard deviations of the means. *, P < 0.05; **, P < 0.001; ***, P < 0.0001 (for comparisons of M1 and −SS to the appropriate WT control set using Student’s two-tailed t test).
FIG 6
FIG 6
Infectivity defects observed in virions with increased GP/Gag ratios are attributed to impaired reverse transcription but not RNA packaging. (A) WT or FS mutants were expressed in the absence (untreated [black bars]; ANOVA, P < 2 × 10−15) or presence (treated [gray bars]; ANOVA, P < 8 × 10−11) of saquinavir in HEK293T cells either alone or coexpressed with GPOnly at a ratio of 5:1 (WT or FS mutant:GPOnly). Full-length unspliced RNA harvested from virions and cell lysates was extracted and quantified by qRT-PCR. RNA packaging efficiency was determined by comparison of in-virion to in-cell (supernatant/cell) RNA relative to WT. *, P < 0.01 for comparison of each condition to the appropriate WT control set using one-way ANOVA. (B) Pseudotyped virus particles harvested from cells expressing WT or FS reporter viruses either alone or coexpressed with GPOnly were assayed for production of reverse transcription products by infecting HEK293T cells. Infected cells were harvested and probed for early RT (black bars; ANOVA, P < 3 × 10−25), late RT (dark gray bars; ANOVA, P < 3 × 10−27), and 2LTR (light gray bars; ANOVA, P < 8 × 10−18) reverse transcription products by qRT-PCR. Virions were probed for p24Gag by immunoblotting to ensure infection of equivalent virions between conditions. Error bars represent the standard deviations of the means. *, P <P 0.001 for comparison of each condition to the appropriate WT control set using one-way ANOVA.
FIG 7
FIG 7
Summary of findings. (A) Compartmentalized cis Gag-GP interactions promote efficient cis delivery of GP to virions. PRF element mutant M1 exhibits ∼3-fold-greater frameshifting than WT virus, resulting in levels of GP incorporation efficiency that can never be achieved by GP expressed in trans (i.e., from a separate gag-pol mRNA). (B) Our data also reinforce that GP is enriched in virions relative to cell lysates and show that HIV-1 will accommodate only an ∼2-fold increase in GP incorporation. Accordingly, the virus has evolved a mechanism to restrict excessive incorporation of GP into virions, likely providing it with a means to buffer per-virion infectivity. (C) M1 particles are less infectious, at least in part because overframeshifting results in premature Gag and GP proteolytic processing in budding virions and impairs the process of reverse transcription.
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