HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA

Abstract

HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection.

Keywords: Cellular factor; Chromosome region maintenance 1; HIV-1; Nef-associated factor 1; Nuclear export; human immunodeficiency virus (HIV); microbiology; molecular cell biology; virology; virus.

FIGURE 1.

Naf1 expression is required for efficient HIV-1 production. A, endogenous Naf1 expression in HEK293T cells was knocked down with specific shRNA and detected by immunoblotting. B and C, knock-down of Naf1 significantly suppresses HIV-1 production in comparison to off-target control in HEK293T cells transfected with HIV-1-based expression vector pH131 (B), or infected with HIV-H131/VSV-G vector (C), and HIV-1 production was quantified by measuring p24^gag amount 24 h later in cell culture supernatants and cell lysates. D and E, Naf1 overexpression enhances HIV-1 production. HEK293T cells were transfected with pCMV-Myc-Naf1, and Naf1 expression was detected by immunoblotting (D). In Naf1-overexpressed cells, viral p24 production was increased (E). F and G, Naf1 complementation in stable Naf1-knocking-down HEK293T cells restores HIV-1 production. Naf1 expression was complemented in stable Naf1-knocking-down HEK293T cells by transfected with pCMV-Myc-Naf1 for 24 h (F), and cells were transfected with the pHIV/H131 plasmid for additional 24 h and viral production was quantified by p24^gag assay (G). GAPDH was used as a loading control (A, D, F). One representative of at least four independent repeats for each result is shown. The results were analyzed by the paired Student's t test; **, p < 0.01 and ***, p < 0.001 were considered statistically significant.

FIGURE 2.

Naf1 expression promotes nuclear export of unspliced HIV-1 gag mRNA. A, effect of Naf1 on HIV-1 transcription. HEK293T cells were transfected with pCMV-Myc-Naf1 or empty vector and pHIV/H131 for 24 h. Cells were harvested at the indicated times post-transfection, and total mRNAs were isolated, and the levels of HIV-1 gag mRNA were quantified with qRT-PCR and normalized to β-actin mRNA. B, scheme for detection of cytoplasmic/nuclear mRNA. Cells were collected for isolation of cytoplasmic and nuclear mRNA, and the relative ratios of cytoplasmic and nuclear mRNA (unspliced gag mRNA or multi-spliced tat-rev mRNA) were analyzed by qRT-PCR. C, U6 snRNA and 18S rRNA was used as nuclear or cytoplasmic RNA control, respectively. C: cytoplasm, N: nucleus. D, Naf1 overexpression enhances the nuclear export of HIV-1 unspliced gag mRNA. Naf1-overexpressing or parental (pCMV-Myc vector) HEK293T cells were transfected with HIV/H131 for 24 h, then cells were collected and total mRNA were isolated, and HIV-1 gag mRNA were quantified as above, the relative cytoplasmic and nuclear distribution of HIV-1 mRNA were calculated, and 4 repeats were summarized. The horizontal lines in the dot plot (low panels) indicate the mean with S.D. E, Naf1 knock-down decreases the nuclear export of HIV-1 unspliced gag mRNA. The Naf1-stable-knock-down HEK293T or off-target cells were infected with the HIV-H131/VSV-G vector for 24 h, and the cytoplasmic/nuclear distribution of HIV-1 mRNA was monitored and the relative values were calculated. ***, p < 0.001 was considered significant as determined by the paired Student's t test.

FIGURE 3.

Naf1 association with CRM1 is required for augmenting HIV-1 production. A and B, Naf1 associates with CRM1. HEK293T cells were co-transfected with plasmids expressing Naf1-Flag and Rev-HA (A), or CRM1-Flag and Myc-Naf1 or Rev-HA (B), the whole cells lysates were prepared, immunoprecipitations were performed with anti-Flag coated magnetic beads, and immunoblotting was performed with indicated specific antibodies. C and D, LMB treatment impairs Naf1-mediated enhancement of HIV-1 Gag production. HEK293T cells were transfected with pCMV-Myc-Naf1 or empty vector and pHIV/H131 plasmid for 18 h. LMB (20 nm) or media were added for an additional 6 h in the cells. Viral production was quantified as above and the relative fold enhancement was calculated (C), and the summary of 4 repeat experiments (individual dots) was presented (D). E, LMB treatment does not have cytotoxicity. Transfected HEK293T cells were incubated with various concentrations of LMB for 6 h, and the LMB cytotoxicity was assessed with MTT method. Cell viability (% of untreated control) was calculated. One representative from three independent repeats is shown. F–H, CRM1 knock-down attenuates Naf1-mediated enhancement of HIV-1 Gag production. HEK293T cells were transfected with CRM1 specific shRNAs or off-target control for 24 h, then cells were transfected with pCMV-Myc-Naf1 or empty vector and pHIV/H131 plasmid for an additional 24 h. The viral production was quantified as above and the relative fold enhancement was calculated (F), and the summary of 4 repeats (individual dots) was presented (G). The CRM1 knock-down was confirmed by immunoblotting (H). I—K, CRM1 overexpression enhances Naf1 function. HEK293T cells were transfected with pcDNA3.1-CRM1-HA for 24 h, then cells were transfected with the pHIV/H131 plasmid for an additional 24 h (I), and viral productions were quantified as above and the relative fold enhancement was calculated (J), and the summary of 4 repeats (individual dots) was presented (K). *, p < 0.05 was considered significant as determined by the paired Student's t test.

FIGURE 4.

The shuttling of Naf1 between the nucleus and the cytoplasm is required for promoting HIV-1 production. A, Naf1 distribution observed with confocal microscopy. HEK293T cells were transfected with peGFP-Naf1 expression plasmid for 24 h, and cells were observed with confocal microscopy. Nuclei were indicated with Hoechst 33258 (blue) stain. B, Naf1 distribution investigated by Western blotting. HEK293T cells were transfected with pCMV-Myc-Naf1 for 24 h, and the cytoplasmic (cyto.) and nuclear (nuc.) components were fractionated for immunoblotting. C, amino acids sequence of Naf1 NESs, NLS, and mutations. D, expression of Naf1 and its mutation detected with immunoblotting. E, distribution of Naf1 and its NLS mutant detected with immunoblotting. F, disruption of NESs or NLS of Naf1 attenuates the enhancement for HIV-1 production. HEK293T cells were transfected with wild type pCMV-Myc-Naf1 or different mutants for 24 h, then cells were further transfected with HIV/H131 plasmid for an additional 24 h, and HIV-1 production was measured by detecting p24^gag amounts in culture medium and cell lysates. *, p < 0.05 and **, p < 0.01 are considered significant difference as determined by paired Student's t test.