Direct participation of Sam68, the 68-kilodalton Src-associated protein in mitosis, in the CRM1-mediated Rev nuclear export pathway

Abstract

Human immunodeficiency virus type 1 (HIV-1) replication requires efficient nuclear export of incompletely spliced and unspliced HIV-1 mRNA transcripts, which is achieved by Rev expression at an early stage of the viral life cycle. We have recently shown that expression of Sam68, the 68-kDa Src-associated protein in mitosis, is able to alleviate Rev function block in astrocytes by promoting Rev nuclear export. In the present study, we utilized an antisense RNA expression strategy to down-modulate constitutive Sam68 expression and examined its effect on Rev function, HIV-1 gene expression, and viral replication. These results showed that down-modulation of constitutive Sam68 expression markedly inhibited HIV-1 production in 293T cells and viral replication in T lymphocytes such as Jurkat and CEM cells, as well as human peripheral blood mononuclear cells (PBMCs). Rev-dependent in trans complementation and reporter gene assays further demonstrated that inhibition of HIV-1 gene expression by Sam68 down-modulation was due to impeded Rev activity. Moreover, digital fluorescence microscopic imaging revealed that down-modulation of Sam68 expression caused exclusive nuclear retention and colocalization of both Rev and CRM1. Taken together, these data suggest that adequate Sam68 expression is required for Rev function and, thereby, for HIV-1 gene expression and viral replication, and they support the notion that Sam68 is directly involved in the CRM1-mediated Rev nuclear export pathway.

FIG. 1.

Inhibition of HIV-1 production by down-modulation Sam68 expression. (a) 293T cells were transfected with one of two anti-Sam68 antisense RNA constructs (6 μg), one containing a longer Sam68 cDNA (1.7 kb) in a reverse orientation (pAs-Sam68a) and the other containing only the 1.3-kb open reading frame of Sam68 cDNA in a reverse orientation (pAs-Sam68b), and were harvested 48 h after transfection for Sam68 expression. Sam68 expression was analyzed by Western blotting. pcDNA3 was also included as a transfection control. Rel., relative levels of protein expression determined by densitometric scanning of the blots using the housekeeping gene actin as an internal standard. (b) 293T cells were transfected with 1.5 μg of HIV-1 NL4-3 proviral DNA with 6 μg of either pcDNA3 (○), pAs-Sam68a (▵), or pAs-Sam68b DNA (⋄). HIV-1 production was determined by measuring the RTase activity of the cell culture supernatant at the time points indicated. (c) 293T cells were transfected with 1.5 μg of HIV-1 NL4-3 proviral DNA with 0 (○), 2 (•), 4 (⋄), or 6 (♦) μg of pAs-Sam68a DNA. pcDNA3 was used to equalize the total amount of DNA transfected among all transfections. HIV-1 production was determined as described above.

FIG. 2.

Inhibition of HIV replication by down-modulation of Sam68 expression. (a and b) pMX.As-Sam68, containing the longer (1.7-kb) Sam68 cDNA in a reverse orientation, was transfected into BOS23 packaging cells by the standard calcium phosphate precipitation method (52), and the culture supernatant was collected 48 h after transfection as virus stocks. Jurkat cells (diamonds), CEM cells (circles), and human PBMCs (triangles) were first transduced with MX viruses (open symbols) or MX.As-Sam68 viruses (solid symbols) for 2 h, and then the free viruses were washed off with fresh cell culture medium. MX virus-transduced cells were immediately infected with HIV-1 NL4-3 with an RTase activity of 20,000 cpm (a) or with an equivalent amount of MLV BV2 (b) for an additional 2 h; then the free viruses were washed off with fresh cell culture medium again, and cells were allowed to grow at 37°C under 5% CO₂. (c) Inhibition of HIV-1 replication is cell autonomous. Jurkat cells were transduced either with pMX viruses (solid bars) or pMX.As-Sam68 viruses (hatched bars) or without MX viruses (mock transduction) (open bars) as described above. The cell culture supernatant was collected 48 h after transduction and added to Jurkat cells infected with HIV-1 NL4-3. HIV-1 replication was determined by measuring the RTase activity of the cell culture supernatant (0.5 ml) at the time points indicated.

FIG. 3.

Inhibition of Rev activity by down-modulation of Sam68 expression. 293T cells were transfected with 0.8 μg of HIV.Δenv alone or HIV.ΔenvΔrev alone, or with different expression plasmids as indicated. The amounts of DNA transfected were as follows: 0.2 μg (+) or 0.4 μg (++) of Rev; 3 μg (+) or 6 μg (++) of As-Sam68a. pcDNA3 was used to equalize the total amount of DNA transfected among all transfections. HIV-1 production was monitored by measurement of RTase activity.

FIG. 4.

Preferential inhibitory effect of Sam68 down-regulation on Rev activity. 293T cells were transfected with 0.5 μg of either RRE-CAT (a), CTE-CAT (b), PRE-CAT (c), or SV2-CAT (d), either alone or in combination with 0.2 μg of Rev and/or 3 μg of As-Sam68a. pcDNA3 was used to equalize the total amount of DNA transfected among all transfections. pCMVβGal was included to normalize variations in transfection efficiency among all transfections. Cells were harvested 48 h after transfection and assayed for CAT activity as previously described (23, 24). CAT activity was determined to be in the linear range.

FIG. 5.

Inhibition of Sam68 expression-induced Rev nuclear export by LMB treatment. 293T cells were transfected with either Rev.GFP, CRM1.RFP, or Sam68.RFP (a) or with both Rev.GFP and Sam68.RFP (b). Transfected cells were first treated with 2 μg of ATD/ml (a) or 10 ng of LMB/ml (b) 48 h after transfection, as indicated, and then fixed and stained with 1 ng of DAPI/ml for the nucleus. Image capturing and analysis were performed as described elsewhere (35).

FIG. 6.

Inhibition of CRM1-mediated Rev nuclear export by Sam68 down-modulation. 293T cells were transfected with the Rev.GFP and CRM1.RFP expression plasmids, either alone or in combination with the anti-Sam68 antisense RNA expression vector AS-Sam68a. pcDNA3 was used to equalize the total amount of DNA transfected among all transfections. LMB treatment, DAPI staining, and image capturing and analysis were performed as described above.

FIG. 7.

Proposed model for Sam68 function in CRM1-mediated Rev nuclear export. Formation of a quadruple complex consisting of Rev, RRE-containing RNA, CRM1, and RanGTP occurs in the nucleus, possibly independently of Sam68. Sam68 then associates with the complex via direct binding to Rev and transports the complex to the NPC, followed by docking onto NPC through CRM1 interaction with nucleoporins. Translocation of Rev, CRM1, and RRE-containing RNAs into the cytoplasm leads to the release of Sam68 into the nucleus.