siRNA targeting the promoter region of SIVmac251 inhibits viral replication in MAGIC-5 cells. (A) 5′LTR sequence of SIVmac251 targeted by siRNAs; si2A and si2A-Scrambled control (si2A-Sc) are shown under the green bar at −164 bp. The NF-κB binding motif is shown above the blue bar at −49 bp. Nucleotide numbering is relative to the transcription start site. (B) The viral reverse transcriptase activity in culture supernatant, measured 6 days after virus infection of MAGIC-5 cells and transfection with FLAG-tagged Ago1 or without FLAG-tagged Ago1- and SIV-specific siRNAs; si2A and siGag, and specificity control, scrambled siRNA (si2A-Sc) or Lipofectamine2000 alone (mock) *P ≤ 0.02, **P = 0.002, *** P = 0.008; NS, not significant; RT, reverse transcriptase.

Immunofluorescent analysis of SIV promoter-targeted siRNA and Ago1 in MAGIC-5 cells. (A) Mock-infected cells co-transfected with Ago1 and si2A. (B) SIV-infected cells co-transfected with Ago1 and si2A. (C) SIV-infected cells co-transfected with Ago1 and scrambled siRNA. siRNAs (si2A and si2A-Sc) labeled with AF555 show red fluorescence, FLAG-tagged epitope representing Ago1 is labeled with AF647 (pseudocolored magenta), Lamin B labeled with AF488 (green fluorescence) indicates nuclear envelope and DAPI nuclear stain is pseudocolored blue. Arrows indicate nuclear localization in SIV-infected cells. Magnification 63×, scale bars indicate 5 μm. Images are representative of three experiments. (D) Pearson's correlation coefficient values are shown for siRNA and DAPI (left panel), Ago1 and DAPI (middle panel) and siRNA and Ago1 (right panel). **P = <0.008.

Immunofluorescent analysis of SIV promoter-targeted siRNA and Ago2 in MAGIC-5 cells. (A) Mock-infected cells co-transfected with Ago2 and si2A. (B) SIV-infected cells co-transfected with Ago2 and si2A. (C) SIV-infected cells co-transfected with Ago2 and scrambled siRNA. siRNAs (si2A and si2A-Sc) labeled with AF555 show red fluorescence, FLAG-tagged epitope representing Ago2 is labeled with AF647 (pseudocolored magenta), Lamin B labeled with AF488 (green fluorescence) indicates nuclear envelope and DAPI nuclear stain is pseudocolored blue. Arrows indicate nuclear localization in SIV-infected cells. Magnification x63, scale bars indicate 5 μm. Images are representative of three experiments. (D) Pearson's correlation coefficient values are shown for siRNA and DAPI (upper left panel), Ago2 and DAPI (upper middle panel), siRNA and Ago2 (upper right panel), siRNA and LaminB (lower left panel) and Ago2 and LaminB (lower right panel). *P ≤ 0.03. **P ≤ 0.008.

siRNA targeting the promoter region of HIV-1 inhibits viral replication in HeLa T4+ cells. (A) 5′LTR sequence of HIV_SF162 and HIV_LAV targeted by siRNAs; siPromA, siPromA-Sc and siPromA-M2 control are highlighted. Nucleotide numbering is relative to the transcription start site. (B) Viral reverse transcriptase activity in culture supernatant, measured 5 days after infection of HeLa T4+ cells and transfection with siRNA alone, FLAG-tagged Ago1 or Ago2 and HIV specific siRNA; siPromA and specificity controls; siPromA-Sc and siPromA-M2 or Lipofectamine2000 alone (mock) *P = < 0.02, **P = 0.002, ***P = 0.008.

Immunofluorescent analysis of HIV-1 promoter-targeted siRNA and Ago1 in HeLa cells. (A) Mock-infected cells co-transfected with Ago1 and siPromA. (B) HIV_SF162-infected cells co-transfected with Ago1 and siPromA. (C) HIV_SF162-infected cells co-transfected with Ago1 and scrambled siRNA. siRNAs (siPromA and siPromA-Sc) labeled with AF555 show red fluorescence, FLAG-tagged epitope representing Ago1 is labeled with AF647 (pseudocolored magenta), Lamin B labeled with AF488 (green fluorescence) indicates nuclear envelope and DAPI nuclear stain is pseudocolored blue. Arrows indicate nuclear localization in HIV_SF162-infected cells. Magnification 63×, bars represent 5 μm. Images are representative of three experiments. (D) Pearson's correlation coefficient values are shown for siPromA and DAPI (left panel), Ago1 and DAPI (middle panel) and siPromA and Ago1 (right panel). **P = 0.002.

Immunofluorescent analysis of HIV-1 promoter-targeted siRNA and Ago2 in HeLa cells. (A) Mock-infected cells co-transfected with Ago2 and siPromA. (B) HIV_SF162-infected cells co-transfected with Ago2 and siPromA. (C) HIV_SF162-infected cells co-transfected with Ago2 and scrambled siRNA. siRNAs (siPromA and siPromA-Sc) labeled with AF555 show red fluorescence, FLAG-tagged epitope representing Ago2 is labeled with AF647 (pseudocolored magenta), Lamin B labeled with AF488 (green fluorescence) indicates nuclear envelope and DAPI nuclear stain is pseudocolored blue. Arrows indicate nuclear localization in HIV_SF162-infected cells. Magnification 63×, bars represent 5 μm. Images are representative of three experiments. (D) Pearson's correlation coefficient values are shown for siPromA and DAPI (upper left panel), Ago2 and DAPI (upper middle panel), siPromA and Ago2 (upper-right panel), siPromA and LaminB (lower left panel) and Ago2 and LaminB (lower right panel). NS, not significant. *P = 0.01, **P = 0.002.

Immunofluorescent analysis of HIV-1 promoter-targeted siRNA and F-actin in HeLa cells. (A) Mock-infected cells co-transfected with Ago1 and siPromA. (B) HIV-infected cells co-transfected with Ago1 and siPromA. (C) HIV-infected cells co-transfected with Ago1 and scrambled siRNA. siRNAs (siPromA and siPromA-Sc) labeled with AF555 show red fluorescence, F-actin is labeled with phalloidin-AF647 (pseudocolored magenta), Lamin B labeled with AF488 (green fluorescence) indicates nuclear envelope and DAPI nuclear stain is pseudocolored blue. Arrows indicate nuclear localization in HIV-infected cells. Magnification 63×, bars represent 5 μm. Images are representative of three experiments. (D) Pearson's correlation coefficient values are shown for siPromA and DAPI (upper left panel), F-actin and DAPI (upper middle panel), siPromA and F-actin (upper right panel), siPromA and LaminB (lower-left panel) and F-actin and LaminB (lower right panel). NS, not significant. * P = 0.02, **P = 0.002.

Immunofluorescent analysis of Ago1 and F-actin in HIV-1-infected HeLa cells. (A) Mock-infected cells co-transfected with Ago1 and unlabeled siPromA. (B) HIV-infected cells co-transfected with Ago1 and siPromA. (C) HIV-infected cells co-transfected with Ago1 and scrambled siRNA. Ago1 labeled with AF555 shows red fluorescence, F-actin is labeled with phalloidin-AF647 (pseudocolored magenta), Lamin B labeled with AF488 (green fluorescence) indicates nuclear envelope and DAPI nuclear stain is pseudocolored blue. Arrows indicate nuclear localization in HIV-infected cells. Magnification 63×, bars represent 5 μm. Images are representative of three experiments. (D) Pearson's correlation coefficient values are shown for Ago1 and DAPI (upper left panel), F-actin and DAPI (upper middle panel), Ago1 and F-actin (upper right panel), Ago1 and LaminB (lower left panel) and F-actin and LaminB (lower right panel). NS, not significant. *P = 0.02, **P = 0.002. (E) IP analysis of HIV-1 infected cells transfected with FLAG-tagged Ago1 or empty vector and siPromA. Cytoplasmic and nuclear fractions applied to IP are shown in the left panel (input) and proteins that immunoprecipitated with the FLAG-M2 agarose affinity gel are shown in the right panel (IP samples). Samples were probed by western blot with antibodies to β-actin (upper panel), Ago (middle panel) or FLAG-M2 (lower panel). WB, western blot; Frac, fraction; C, cytoplasmic; N, nuclear; Ctrl-FLAG, control empty vector.

Immunofluorescent analysis of HIV-1 promoter-targeted siRNA and F-actin in cytochalisin D-treated HeLa cells. (A) HIV-1_SF162 infected cells co-transfected with Ago1 and siPromA, not treated with cytochalisin D (CytD). (B) HIV-1_SF162 infected cells co-transfected with Ago1 and siPromA, not treated with CytD. (C) HIV-1_SF162 infected cells pre-treated with cytochalisinD and co-transfected with Ago1 and siPromA. siRNAs labeled with AF555 show red fluorescence, F-actin is labeled with phalloidin-AF647 (pseudocolored magenta), Lamin B labeled with AF488 (green fluorescence) indicates nuclear envelope and DAPI nuclear stain is pseudocolored blue. Arrows indicate nuclear localized F-actin staining in panel b and punctate cytoplasmic F-actin staining in (C). Magnification 63×, bars represent 5 μm. Images are representative of three experiments. (D) Pearson's correlation coefficient values for CytD treated cultures are shown for siRNA and DAPI (left panel), F-actin and DAPI (middle panel) and siRNA and F-actin (right panel). NS, not significant; *P = 0.01.

Model of the actin cytoskeleton role in nuclear trafficking of mammalian RITS-like complex components. Our results show that Ago1 and promoter-targeted siRNA co-localize in the nucleus of retrovirus-infected cells as part of the RITS-like complex during TGS, while Ago2 and promoter-targeted siRNAs co-localize in the inner nuclear envelope membrane (inner NM) of retrovirus-infected cells during TGS. Our findings suggest that nuclear delivery of RITS-like complex components, specifically promoter-targeted siRNAs associated with Ago1, involves the cytoskeleton actin filament (F-actin), probably via active transport through the nuclear pore complex (NPC). In response to F-actin translocating into the nucleus, we speculate that monomeric actin (G-actin) levels would increase in the cytoplasm. Shown on the left is a cell actively undergoing transcription, prior to transfected siRNAs associating with Ago protein/s. On the right is a cell undergoing TGS, where the siRNAs have associated with Ago protein/s and are trafficked into the nucleus with the involvement of the actin cytoskeleton.