Retroviral mRNA nuclear export elements regulate protein function and virion assembly

Abstract

Rodent cells are notable for their inability to support normal assembly of HIV particles. In this report, we address possible causes for this defect by considering the hypothesis that mRNA-associated events occurring in the nucleus can regulate the activity of their encoded proteins in the cytoplasm. We show that altering the RNA nuclear export element used by HIV gag-pol mRNA from the Rev response element to the constitutive transport element restores both the trafficking of Gag to cellular membranes and efficient HIV assembly in murine cells. These results suggest that two phases of the HIV life cycle, RNA export and capsid assembly, that have hitherto been regarded as distinct are, in fact, linked. Thus, protein function and fate may depend upon the full and precise history of its encoding mRNA.

Figure 1

HIV Gag-Pol constructs. GPV-Δ, GPV-RRE, GPV-1 × CTE and GPV-4 × CTE were cloned into the pcDNA 3.1 vector with a CMV-IE promoter (CMV) and a bovine growth hormone polyadenylation signal (BGH pA). Splice donors (SD) and splice acceptors (SA) are marked. GP-RevInd was a codon-optimized Gag-Pol cloned into the pCI-Neo vector with the CMV-IE promoter (CMV) and the SV40 polyadenylation signal (SV40 pA) (Kotsopoulou et al, 2000). The vector contained an intron 5′ of the Gag-Pol coding region.

Figure 2

Rev-independent HIV Gag-Pol expression results in efficient Gag processing and budding in murine cells. HeLa (lanes 1 and 2) or 3T3 (lanes 3 and 4) cells were transfected with GPV-RRE+pcRev (lanes 1 and 3) or GP-RevInd+pcDNA 3.1 (lanes 2 and 4) expression vectors and then analyzed at ∼40 h for the presence of Gag proteins in whole-cell lysates and culture media by immunoprecipitation/immunoblot. The p24^Gag content in the media from the 3T3 cells was also quantitated by ELISA, and GPV-RRE-transfected cells (lane 3) produced 0.88 ng/ml and the GP-RevInd-transfected cells (lane 4) produced 12 ng/ml.

Figure 3

HIV Gag processing and budding are restored in murine cells by 4 × CTE/NXF1-mediated RNA export. (A, B) The 4 × CTE confers efficient budding in HeLa (A) and 3T3 (B) cells. Cells were transfected with the indicated GPV constructs and Gag proteins analyzed as in Figure 2. The 3T3 samples were examined further by ELISA (B, lower panel). (C) ³H myristylate labeling of Gag in 3T3 cells transfected with GPV-RRE+pcRev (lane 2) or GPV-4 × CTE (lane 3); lane 1 represents mock-transfected cells. Gag proteins were immunoprecipitated from cell lysates, resolved by SDS–PAGE and visualized by autoradiography. (D) The 4 × CTE enhances budding of the HIV Gag-Pol with the MLV MA membrane targeting domain. 3T3 cells transfected with GPV vectors based on wild-type NL4-3 or NL4-3 with the HIV MA domain replaced by the MLV MAp12 domains. Filtered media from the tranfections were analyzed by p24^Gag ELISA.

Figure 4

The 4 × CTE RNA export element does not enhance budding in murine cells by increasing intracellular Gag concentration or by altering splicing. (A) The induction of Gag budding from 3T3 cells transfected with GPV-4 × CTE (lanes 1–3) versus GPV-RRE+pcRev (lane 4) is not due to variations in intracellular Gag concentration. Cells were transfected either with a titration of GPV-4 × CTE (100%=2 μg pGPV-4 × CTE+1 μg pcDNA3.1, total DNA levels were maintained by the addition of pcDNA3.1) or 2 μg of GPV-RRE+1 μg pcRev, and the Gag proteins were analyzed as in Figure 1A. (B) The RRE and 4 × CTE RNA export elements do not lead to differences in splicing. Cytoplasmic RNA was isolated from 3T3 cells transfected with GPV-RRE or GPV-4 × CTE and analyzed by Northern blot with a probe specific for the gag gene. The difference in transcript sizes is due to the 441 bp difference in the length of the RRE compared to the 4 × CTE.

Figure 5

4 × CTE/NXF1-mediated nuclear export promotes the association of Gag with the plasma membrane. (A) RNA export elements modulate Gag's ability to associate with membranes using a membrane flotation assay. 3T3 cells were transfected with GPV-RRE+pcRev (left panels) or GPV-4 × CTE (right panels) and the resulting postnuclear supernatants were analyzed using a membrane flotation assay. Following fractionation, all samples were examined for Gag content by immunoprecipitation/immunoblot and for transferrin receptor (TR) by immunoblot. The smaller TR bands (marked with ^*) are degradation products, but as the antibody used is a monoclonal to the cytoplasmic tail, these are additive with the full-length 95 kDa protein. Fractions 3 and 4 contain membrane-associated proteins and, potentially, demembranated immature viral cores. (B) Indirect immunofluorescence shows that RNA export elements modulate Gag's ability to be targeted to the plasma membrane. Subconfluent mouse 3T3 cells were seeded on glass coverslips and transfected with GPV-RRE (panels (i) and (ii)) or GPV-4 × CTE (panels (iii) and (iv)). At 20 h post-transfection, the cells were fixed and stained with rabbit polyclonal anti-MA antibody.

Figure 6

The 4 × CTE does not enhance Gag budding in murine cells when the RNA is directly expressed in the cytoplasm. 3T3 cells were infected with vaccinia viruses expressing GPV-Δ, GPV-RRE or GPV-4 × CTE. At 48 h after infection, lysates and media were harvested and the Gag proteins analyzed as in Figure 2. The relative levels of p24^Gag in the media represent the mean of three independent experiments normalized to the vvGPV-Δ samples (mean value of 1.81 μg/ml) with error bars representing standard deviation. All three viruses expressed similar amounts of intracellular Gag (not shown).