HIV-1 requires Staufen1 to dissociate stress granules and to produce infectious viral particles

Abstract

The human immunodeficiency virus type 1 (HIV-1) genomic RNA (vRNA) has two major fates during viral replication: to serve as the template for the major structural and enzymatic proteins, or to be encapsidated and packaged into assembling virions to serve as the genomic vRNA in budding viruses. The dynamic balance between vRNA translation and encapsidation is mediated by numerous host proteins, including Staufen1. During HIV-1 infection, HIV-1 recruits Staufen1 to assemble a distinct ribonucleoprotein complex promoting vRNA encapsidation and viral assembly. Staufen1 also rescues vRNA translation and gene expression during conditions of cellular stress. In this work, we utilized novel Staufen1^-/- gene-edited cells to further characterize the contribution of Staufen1 in HIV-1 replication. We observed a marked deficiency in the ability of HIV-1 to dissociate stress granules (SGs) in Staufen1-deficient cells and remarkably, the vRNA repositioned to SGs. These phenotypes were rescued by Staufen1 expression in trans or in cis, but not by a dsRBD-binding mutant, Staufen1F135A. The mistrafficking of the vRNA in these Staufen1^-/- cells was also accompanied by a dramatic decrease in viral production and infectivity. This work provides novel insight into the mechanisms by which HIV-1 uses Staufen1 to ensure optimal vRNA translation and trafficking, supporting an integral role for Staufen1 in the HIV-1 life cycle, positioning it as an attractive target for next-generation antiretroviral agents.

Keywords: HIV-1; Staufen1; stress granules; stress response to infection; vRNA trafficking; viral genomic RNA (vRNA).

FIGURE 1.

HIV-1 has impaired ability to dissociate Ars-induced SGs in SKO cells and is rescued by Staufen1 in trans and in cis. (A) HCT116 cells were transfected with pcDNA3.1 or proviral HIV-1 construct, pNL4.3. Twenty-four hours later, cells were either mock-treated or treated with 500 µM Ars for 1 h and then p24 (green) and TIAR1 (red) were detected by indirect immunofluorescence. Nuclei were stained with DAPI (blue). Scale bars are 10 µm. (B) Staufen1 gene-edited SKO cells were transfected with pcDNA3.1 (Mock), pNL4.3, or pNL4.3 and a Staufen1/YFP cDNA expression construct to rescue Staufen1 in trans. Twenty-four hours later, cells were either mock treated or treated with Ars for 1 h and p24 (green) and TIAR1 (red) were detected by indirect immunofluorescence. Nuclei were stained with DAPI (blue). Gag is pseudocolored green; inset shows Staufen1-YFP expression in yellow. Scale bars are 10 µm. (C) HCT116 cells or SKO cells (as indicated) were transfected either with pNL4.3, pNL4.3/Staufen1-HA, or pNL4.3/Staufen1F135A-HA, to rescue with WT or dsRBD3 mutant Staufen1, in cis. Twenty-four hours later, cells were either mock treated or treated with Ars for 1 h, and p24 (green) and eIF3 (pink) were detected by indirect immunofluorescence. Nuclei were stained with DAPI (blue). Scale bars are 10 µm. (D) Quantification of cells containing SGs from panels A–C. Error bars represent the standard deviation from three independent experiments with at least 100 cells counted per treatment. Asterisks represent statistically significant differences between groups (one-way ANOVA: [**] P ≤ 0.01; [****] P ≤ 0.001).

FIGURE 2.

HIV-1 genomic RNA (vRNA) is sequestered in Ars-induced SGs in SKO cells. (A) HCT116 cells were transfected with pcDNA3.1 or pNL4.3. Twenty-four hours later, cells were either mock treated or treated with Ars for 1 h, and then HIV-1 genomic RNA (vRNA; red) and TIAR1 (cyan) were identified by coupled fluorescence in situ hybridization/immunofluorescence. Nuclei were stained with DAPI (blue). Scale bars are 10 µm. (B) SKO cells were transfected with pcDNA3.1, pNL4.3, or pNL4.3.-Staufen1-HA to rescue Staufen1 expression in cis. Twenty-four hours later, cells were either mock treated or treated with Ars for 1 h and then p24 (green) and TIAR1 (cyan) were detected by indirect immunofluorescence. Nuclei were stained with DAPI (blue). Scale bars are 10 µm.

FIGURE 3.

SKO cells exhibit impaired intracellular vRNA expression. (A) HCT116 and SKO cells were transfected with either pNL4.3 or pNL4.3-Staufen1-HA, to rescue Staufen1 in cis. Twenty-four hours later, cell lysates were subjected to SDS-PAGE, immunoblotted, and probed to investigate eIF2α phosphorylation and Gag expression. (B) Densitometric quantification of phosphor(P)-eIF2α was determined by ImageJ analysis. Values presented in the graph are normalized against the total amount of eIF2α in the cell lysate and represent fold change with the HCT116 pNL4.3-transfected cells being arbitrarily set to one. Error bars represent the standard error of the mean from three independent experiments. Asterisks represent statistically significant differences between groups (one-way ANOVA: [**] P ≤ 0.01; [***] P ≤ 0.005). (C) Densitometry quantifications of Gag levels were determined by ImageJ analysis. Values presented in the graph are normalized against the total amount of Gag in the cell lysate and represent fold change with the HCT116 pNL4.3-transfected cells being arbitrarily set to one. Error bars represent the standard error of the mean from three independent experiments. Asterisks represent statistically significant difference between groups (one-way ANOVA: [**] P ≤ 0.01). (D) HIV-1 genomic RNA, vRNA, and levels from cells transfected as described above were measured by RT-PCR and normalized to the HCT116 pNL4.3-transfected condition that was arbitrarily set to one. Error bars represent the standard deviation from three independent experiments. ns, no statistical differences between the means.

FIGURE 4.

The viruses generated from SKO cells have impaired infectivity. HCT116 and SKO cells were transfected with either pNL4.3 or pNL4.3.-Staufen1-HA to rescue Staufen1 in cis. The amount of p24 in the supernatant of transfected cells was measured by p24 ELISA. Error bars represent the standard deviation from three independent experiments. Asterisks represent statistically significant difference between groups (one-way ANOVA: [****] P ≤ 0.001). (B) vRNA levels from cells transfected as described above were measured by RT-PCR and normalized to the amount of virus in the supernatant as measured by p24 ELISA. Error bars represent the standard deviation from three independent experiments. Asterisks represent statistically significant differences between groups (one-way ANOVA: [**] P ≤ 0.01). (C) The infectivity of the virus in the supernatants of cells transfected as described above was quantified by X-Gal staining assay and normalized to the amount of virus in the supernatant as measured by p24 ELISA. Error bars represent the standard deviation from three independent experiments. Asterisks represent statistically significant difference between groups (one-way ANOVA: [**] P ≤ 0.01).