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. 2022 Feb 28;50(4):2302-2318.
doi: 10.1093/nar/gkac062.

Epitranscriptomic regulation of HIV-1 full-length RNA packaging

Camila Pereira-Montecinos  1   2 , Daniela Toro-Ascuy  1   2 , Catarina Ananías-Sáez  1   2 , Aracelly Gaete-Argel  1   2 , Cecilia Rojas-Fuentes  1   2 , Sebastián Riquelme-Barrios  1   2 , Bárbara Rojas-Araya  1   2 , Francisco García-de-Gracia  1   2 , Paulina Aguilera-Cortés  1   2 , Jonás Chnaiderman  1 , Mónica L Acevedo  1 , Fernando Valiente-Echeverría  1   2 , Ricardo Soto-Rifo  1   2
Affiliations

Epitranscriptomic regulation of HIV-1 full-length RNA packaging

Camila Pereira-Montecinos et al. Nucleic Acids Res. .

Erratum in

Abstract

During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that deposition of N6-methyladenosine (m6A) regulates full-length RNA packaging. While m6A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation promoted the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and contributes to full-length RNA demethylation. We further identified two highly conserved adenosines within the 5'-UTR that have a crucial functional role in m6A methylation and packaging of the full-length RNA. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the HIV-1 full-length RNA molecules that will be used as viral genomes.

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Figures

Figure 1.
Figure 1.
The presence of m6A within the full-length RNA favors Gag synthesis but interferes with packaging. HEK293T cells were transfected with pNL4.3 and pCMV-VSVg together with pCDNA-Flag-METTL3 and pCDNA-Flag-METTL14 or pCDNA-d2EGFP as a control. (A) At 24 hpt cells extracts were used to detect Gag, Flag-METTL3 and Flag-METTL14 by Western blot. GAPDH was used as a loading control (left panel). In parallel, cells extracts were used to perform RNA extraction and the full-length RNA was quantified by RT-qPCR (right panel). Intracellular full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (*P< 0.05, t-test). (B) Supernatants from cell cultures in (A) were filtered and viral particles were purified by ultracentrifugation. The level of CAp24 was quantified by an anti-CAp24 ELISA. The level of CAp24 was normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (ns; non-significant, t-test) (left panel). Viral particles purified were used to perform RNA extraction and the packaged full-length RNA from CAp24 equivalents was quantified by RT-qPCR. Packaged full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (right panel). (C) METTL3 knockdown HEK293T cells were transfected with pNL4.3 and pCMV-VSVg. At 24 hpt cells extracts were used to detect Gag, METTL3 and GAPDH by Western blot. (D) Supernatant from cell cultures were filtered and viral particles were purified by ultracentrifugation. The level of CAp24 was quantified by an anti-CAp24 ELISA and the packaged full-length RNA from CAp24 equivalents was quantified by RT-qPCR. Packaged full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments. (*P< 0.05; ***P < 0.001, t-test). (E) HeLa cells were transfected with pNL4.3, pCMV-VSVg and pCDNA-Flag-METTL3. At 24 hpt, the interaction between full-length RNA and Flag-tagged METTL3 was analyzed by ISH-PLA as described in the Materials and Methods section. Red dots indicate the interactions between the full-length RNA and Flag-METTL3. Scale bar 10 mm. A quantification of the red dots in the nucleus (co-localizing with the DAPI staining) and the cytoplasm is presented on the right (****P< 0.0001, Mann–Whitney test). (F) HeLa cells were transfected with pNL4.3, pCMV-VSVg, pCDNA-Flag-METTL3 and pCDNA-Flag-METTL14. At 24 hpt, the interaction between the full-length RNA and Gag was analyzed by ISH-PLA as described in the Materials and Methods section. Red dots indicate the interactions between the full-length RNA and Gag. Scale bar 10 mm. A quantification of the red dots in both conditions is presented on the right (****P< 0.0001, Mann–Whitney test).
Figure 2.
Figure 2.
Packaged full-length RNA lacks m6A residues at the 5′-UTR. (A) HEK293T cells were transfected with pNL4.3 and pCMV-VSVg. Intracellular polyA RNA or viral particle-associated RNA was extracted at 24 hpt, fragmented and used for MeRIP-seq as described in Materials and Methods section. Peak calling plots for the intracellular (left) and packaged (right) full-length RNA are shown. The drop in the peak near the beginning of the Nef coding region corresponds to the site where the EGFP-Puro cassette is inserted. (B) SupT1 cells were infected with HIV-1 NL4.3 pseudotyped with VSVg. At 24 hpi, supernatants were filtered, and viral particles were concentrated by ultracentrifugation. The RNA from cells and viral particles were used to perform a MeRIP-RT-qPCR specific for the 5′-UTR as described in the Materials and Methods section.
Figure 3.
Figure 3.
Adenosine residues 198 and 242 are key for HIV-1 full-length RNA packaging. (A) HEK293T cells were transfected with pNL4.3 wild type, pNL4.3 ΔA198, pNL4.3 ΔA242 or pNL4.3 ΔA198/ΔA242 together with pCMV-VSVg. At 24 hpt, cells extracts were used to detect Gag by Western blot. Actin was used as a loading control (left panel). In parallel, cells extracts were used to perform RNA extraction and the full-length RNA was quantified by RT-qPCR (right panel) (*P< 0.05; ns; non-significant, t-test). (B) The supernatant from (A) was filtered and concentrated viral particles were used to perform anti-CAp24 ELISA and RNA extraction for RT-qPCR analysis. The levels of CAp24 and the packaged full-length RNA (per CAp24 equivalents) were normalized to the wild type provirus (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (*P< 0.05; ns; non-significant, t-test). (C) HEK293T cells were transfected with pNL4.3 wild type or pNL4.3 ΔA198/ΔA242 together with pCMV-VSVg. At 24 hpt the RNA from cells was used to perform a MeRIP-RT-qPCR as described in the Materials and Methods section (*P< 0.05; n.s, t-test). (D) HEK293T cells were transfected with pNL4.3 wild type or pNL4.3 ΔA198/ΔA242 together with pCMV-VSVg, pCDNA-Flag-METTL3 and pCDNA-Flag-METTL14 or pCDNA-d2EGFP as a control. At 24 hpt cells extracts were used to detect Gag, Flag-METTL3 and Flag-METTL14 by Western blot. GAPDH was used as a loading control (left panel). In parallel, cells extracts were used to perform RNA extraction and the full-length RNA was quantified by RT-qPCR (right panel). Intracellular full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (ns; non-significant, t-test). (E) At 24 hpt supernatants were filtered and viral particles were concentrated by ultracentrifugation. Purified viral particles were used to perform an anti-CAp24 ELISA and RNA extraction and RT-qPCR analysis. The levels of CAp24 and the packaged full-length RNA (per CAp24 equivalents) were normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (*P< 0.05; **P< 0.01; ns, non-significant, t-test).
Figure 4.
Figure 4.
Demethylation by a Gag-FTO complex favors HIV-1 full-length RNA packaging. (A) HEK293T cells were transfected with pNL4.3 and pCMV-VSVg together with pCDNA-3XFlag-FTO or pCDNA-3XFlag-d2EGFP as a control. At 24 hpt cells extracts were used to detect Gag and 3XFlag-FTO by Western blot. GAPDH was used as a loading control (left panel). In parallel, cells extracts were used to perform RNA extraction and the full-length RNA was quantified by RT-qPCR (right panel). Intracellular full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (*P< 0.05, t-test). (B) Supernatants from cell cultures in (A) were filtered and viral particles were concentrated by ultracentrifugation. The level of CAp24 was quantified by an anti-CAp24 ELISA, normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (n.s; non-significant, t-test) (left panel). Viral particles were used to perform RNA extraction and the packaged full-length RNA from CAp24 equivalents was quantified by RT-qPCR. Packaged full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean +/− SD of three independent experiments (**P < 0.01, t-test) (right panel). (C) HeLa cells were co-transfected with pNL4.3, pCMV-VSVg and pCDNA-3XFlag-FTO. At 24 hpt, the interaction between Gag and Flag-tagged FTO was analyzed by PLA as described in Materials and Methods section. Red dots indicate the interactions between Gag and FTO (left panel). Scale bar 10 mm. (D) Three-dimensional reconstitution of the PLA results shown in (C) was performed to determine the subcellular localization of the interaction between Gag and FTO. (E) Quantification of the red dots in the nucleus (co-localizing with the DAPI staining) and the cytoplasm of 15 cells is presented on the right (****P< 0.0001, Mann–Whitney test). (F) HEK293T cells were transfected with the pNL4.3 wild type, pNL4.3-GagStop or pNL4.3-GagStop together with pCDNA-Gag. At 24 hpt cells extracts were used to detect Gag and GAPDH was used as a loading control. In parallel, cells extracts were used to perform RNA extraction followed by an immunoprecipitation using an anti-m6A antibody (MeRIP-RT-qPCR as described in Materials and Methods section). The full-length RNA from the input was quantified by RT-qPCR (left panel). The full-length RNA from the input (‘A’ fraction) and from the immunoprecipitated material (‘m6A’ fraction) was quantified by RT-qPCR. The m6A/A ratio was normalized to pNL4.3 wild type (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (*P< 0.05, t-test).
Figure 5.
Figure 5.
Inhibition of FTO demethylase activity impacts full-length RNA metabolism and blocks packaging. (A) HEK293T cells were transfected with pNL4.3 and pCMV-VSVg and were treated with MA2 or DMSO as a control. At 24 hpt, cells extracts were used to detect Gag and GAPDH as a loading control (left panel). In parallel, cells extracts were used to perform RNA extraction and the full-length RNA was quantified by RT-qPCR (right panel). The intracellular full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (***P< 0,001, t-test). (B) Supernatants from (A) were filtered, and viral particles were concentrated by ultracentrifugation. The level of CAp24 was quantified by an anti-CAp24 ELISA, normalized to the control (arbitrary set to 100%) and presented as the mean ± SD of three independent experiments (ns; non-significant, t-test) (left panel). Purified viral particles were used to perform an RNA extraction and the packaged full-length RNA from CAp24 equivalents was quantified by RT-qPCR. Packaged full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean +/− SD of three independent experiments (right panel). (C) and (D) The same experiment was performed using pNL4.3 ΔA198/ΔA242 (*P< 0.05, t-test).
Figure 6.
Figure 6.
Working model for the epitranscriptomic regulation of HIV-1 full-length RNA packaging. The HIV-1 full-length RNA is methylated by the METTL3/14 complex in the nucleus (for simplicity, only the presence of m6A on the 5′-UTR is shown). A fraction of the structural protein Gag is imported to the nucleus, interacts with the m6A eraser FTO to promote demethylation of adenosines residues present at the 5′-UTR in a process required for full-length RNA packaging (created with BioRender).
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