7SL RNA packaging into HIV-1 virus like particles is dependent on nucleocapsid. A. Schematic representation of HIV-1 VLP-forming GAG construct and its derivative, Zwt-p6. B. VLPs were produced by 293T cells transfection with GAG or Zwt-p6 constructs in the presence of APOBEC3G, APOBEC3F or mock plasmid as indicated. Intracellular and VLP levels of cytidine deaminases and Gag were measured by Western blotting with HA- and capsid-specific antibodies, respectively. C. 7SL RNA in viral like particles was quantified by real time PCR. A representative experiment out of two is shown.

7SL RNA knockdown does not prevent A3G encapsidation. A. 7SL RNA in cells lines expressing control-shRNA and SRP14-shRNA, was quantified by real time PCR. APOBEC3G or APOBEC3F expressing plasmids were transfected when indicated. Means and standard errors from three independent experiments are shown. B. Production of Vif-defective HIV-1 particles from control-shRNA or SRP14-shRNA cell lines, in the presence of APOBEC3G or APOBEC3F when indicated. Means and standard errors from three independent experiments are shown. C. 7SL RNA, Y3 RNA and 5S RNA measured by real time PCR in Vif defective HIV-1 particles produced from control-shRNA or SRP14-shRNA cell lines, upon transfection of APOBEC3G or APOBEC3F when indicated. Results were normalized to viral genomic RNA. Means and standard errors from three independent experiments are shown. D. Western blot analysis of these viruses and of cytoplasmic extracts of the producer cells, using indicated antibodies, ß-actin and capsid serving as loading controls. E. Infectivity of Vif-defective HIV-1 particles produced from control-shRNA or SRP14-shRNA cell lines, in the presence of APOBEC3G or APOBEC3F when indicated.

APOBEC3G packaging mutant is defective for 7SL and Alu RNAs binding, and it does not inhibit Alu retrotransposition. A. 7SL RNA capture assay as described in figure 1B, transfecting 293T cells with indicated doses of wild-type or W127L APOBEC3G-expresssing plasmid. Top: HA-specific Western blot; middle: standard RT-PCR; bottom: real-time RT-PCR. B. 7SL RNA and endogenous Alu RNAs capture assay as described in figure 1B, transfecting 293T cells with indicated doses of wild-type or W127L APOBEC3G-expresssing plasmid. Top: HA-specific Western blot; bottom: real-time RT-PCR. C. Alu (pAlu pA+ neoTet) and L1 (pJ M101 L1-RP Δneo) expressing plasmids were transfected in HeLa cells in the presence of the indicated cytidine deaminases. Western blot analysis of total cell extracts, with PCNA- and HA-specific antibodies (top). Scoring of G418-resistant colonies (bottom). Means and SE from three representative experiments are shown.

A packaging-defective APOBEC3G mutant. A. Infectivity of Vif-defective HIV-1 particles (expressed in HeLa P4.2 transducing units normalized for RT activity) produced in the presence of wild-type (A3G) or W127L APOBEC3G by transfection of 293T cells. Numbers below columns represent amount of transfected DNA in μg. Representative of five independent experiments. B. Western blot analysis of these particles and of cytoplasmic extracts of the transfected cells, using indicated antibodies, PCNA and capsid serving as loading controls. Although well expressed, APOBEC3GW127L is not packaged into virions. C. APOBEC3GW127L is sensitive to HIV-1 Vif-induced degradation. HIV-1 Vif was co-expressed in 293T cells with either wild type or W127L HA-tagged APOBEC3G, and extracts were analyzed by HA-specific Western blotting. D. HA-specific indirect immunofluorescence analysis of cells expressing HA-tagged versions of wild-type or W127L APOBEC3G. Both proteins localize to the cytoplasm, although the W127L mutant may exhibit a slightly more diffuse distribution.

APOBEC3G binds 7SL RNA. A. Predicted secondary structure of Alu and 7SL RNAs. The latter comprises Alu and S domains. Bases in red are identical between the Alu used in this study (belonging to Alu Sb1 family) and 7SL. For competition experiments (see 2D), the indicated Alu and S stem domains of 7SL RNA were used. B. Indicated HA-tagged cytidine deaminases were immunoprecipitated. The resulting material was analyzed by HA-specific western blot (top) and 7SL RNA-specific standard (middle) or real-time (bottom) RT-PCR. For APOBEC3G, indicated three doses of plasmid were transfected; for all the others, 20 μg of DNA were used. NT, non-transfected. C. Alu RNA competes with 7SL RNA for binding to APOBEC3G. Increasing doses of Alu-Sb1 RNA-expressing plasmid were co-transfected with a fixed amount of APOBEC3G DNA. Upper panel: APOBEC3G in total cellular homogenates in one representative experiment. PCNA was blotted as a loading control. Bottom panel: APOBEC3G was immunoprecipitated and Real Time PCR was used to quantify levels of 7SL RNA in the immunoprecipitates. Means and SE from three independent experiments are shown. D. The Alu domain of 7SL RNA competes with full-length 7SL RNA for APOBEC3G binding. Alu-Sb1 RNA or 7SL Alu and S stem domains-expressing plasmids were co-transfected with APOBEC3G DNA at a 3:1 ratio. Upper panel: APOBEC3G in total cellular homogenates in one representative experiment, PCNA serving as a loading control. Bottom panel: 7SL RNA-specific real time PCR on APOBEC3G-specific immunoprecipitates. Means and SD from two independent experiments are shown.

7SL RNA down regulation by over expression of SRP19 does not inhibit APOBEC3F and 3G encapsidation and antiviral effect. A. Effect of SRP19 overexpression on virus production and infectivity. HIV-1 ΔVif lentiviral vector particles were produced by transient transfection of 293T cells, adding indicated amounts of APOBEC3G, 3F, SRP19 and SRP19 Δ6 plasmids. Virion production (white columns) and infectivity (gray columns) were assessed by measuring reverse transcriptase activity in the supernatant and performing a single round transduction assay, respectively. SRP19 overexpression affects neither HIV particle release nor the antiviral action of APOBEC3G and 3F. Results are representative of three independent experiments. B. SRP19 over expression inhibits 7SL RNA virion incorporation. HIV genomic (white columns) and 7SL RNA (gray columns) levels in virions produced as described in (A) were quantified by real time PCR. Amounts measured in virions produced from cells transfected with ΔVif HIV1 alone were given the arbitrary value of 100. SRP19 overexpression drastically reduces 7SL RNA levels in virions, in spite of having no measurable effect on the intracellular levels of this RNA species (not illustrated). Representative of three independent experiments. C. SRP19 overexpression does not impair APOBEC3F/3G virion incorporation. Western blot analysis of viral and cellular extracts after transient transfection of 293T cells as described in (A), using indicated antibodies. The normal virion incorporation of A3G and A3F corroborates the absence of effect of SRP19 overexpression on the antiviral action of the cytidine deaminases (A).