Fig 1.
RAB11FIP5 interacts with KSHV ORF45.
(A) HEK293T cells were transfected with Flag-ORF45 alone, with HA-RAB11FIP5 alone or with both Flag-ORF45 and HA-RAB11FIP5. Cell lysates were immunoprecipitated with an anti-Flag antibody and were then analyzed by western blotting with the indicated antibodies. (B) HEK293T cells were transfected with Flag-RAB11FIP5 alone, with HA-ORF45 alone or with both Flag-RAB11FIP5 and HA-ORF45. Cell lysates were immunoprecipitated with an anti-Flag antibody and were then analyzed by western blotting with the indicated antibodies. (C) In vitro GST affinity binding assay. Bacterially expressed GST and GST-RAB11FIP5 bound to GST-Sepharose beads were incubated with purified His-tagged ORF45, and the pulled down lysates were immunoblotted with anti-His or anti-GST antibodies. Colocalization of RAB11FIP5 and ORF45 in HeLa cells (D) and HEK293T cells (E). After transfection with Flag-RAB11FIP5 and HA-ORF45, HeLa cells and HEK293T cells were fixed with 4% paraformaldehyde and were then labeled with anti-HA and anti-Flag antibodies. FITC- and Cy3-conjugated secondary antibodies were used to visualize the labeled RAB11FIP5 and ORF45 proteins, respectively. DAPI was used to label cell nuclei.
Fig 2.
The interaction between endogenous RAB11FIP5 and ORF45.
(A) Co-IP of endogenous ORF45 and RAB11FIP5 in KSHV-positive iSLK.RGB cells. Lytic replication of KSHV in the cells was induced by dox, and cell lysates were subjected to immunoprecipitation with the anti-ORF45 antibody or mouse IgG control antibody. Purified proteins, along with input samples, were subjected to western blotting with the indicated antibodies. (B) Co-IP of endogenous ORF45 and RAB11FIP5 in KSHV-positive BCBL1 cells. Lytic replication of KSHV in the cells was induced by VPA, and the cells were treated as described in (A). (C) Endogenous RAB11FIP5 colocalized with endogenous ORF45 in the cytoplasm. BCBL1 cells uninduced (Un) or induced with VPA (In) were fixed and labeled with anti-RAB11FIP5 and anti-ORF45 antibodies and were then incubated with FITC- or Cy3-conjugated secondary antibodies.
Fig 3.
Mapping the interaction domains in ORF45 and RAB11FIP5.
(A) Schematics of the ORF45 truncation mutants, including 115–407 (aa 115-aa 407), 90–407 (aa 90-aa 407), 77–407 (aa 77-aa 407), 19–407 (aa 19- aa 407), 1–332 (aa 1- aa 332), 1–237 (aa 1- aa 237), and 1–114 (aa 1- aa 114), are shown. (B) Defining the RAB11FIP5-interacting domain in ORF45. Co-IP and western blotting of HEK293T cells cotransfected with HA-tagged RAB11FIP5 and a vector expressing the indicated Flag-tagged ORF45 truncations or full-length ORF45. (C) Schematic diagram of ORF45 deletion mutants (Δ237–332 and Δ115–332). (D) Co-IP and western blotting of HEK293T cells cotransfected with HA-RAB11FIP5 and Flag-tagged ORF45 deletion mutants. (E) Schematics of RAB11FIP5 truncation mutants, including 630–653 (aa 630- aa 653), 1–490 (aa 1- aa 490), 490–653 (aa 490- aa 653), and 16–127 (aa 16- aa 127), are shown. (F) Defining the ORF45-interacting domain in RAB11FIP5. Co-IP and western blotting of HEK293T cells cotransfected with HA-tagged ORF45 and the indicated Flag-tagged RAB11FIP5 truncations or full-length RAB11FIP5. An empty vector was used as the negative control. (G) Schematic diagram of a C2 region deletion mutant of RAB11FIP5 (Δ16–127). (H) Co-IP and western blotting of HEK293T cells cotransfected with HA-ORF45 and the Flag-tagged C2 region deletion mutant of RAB11FIP5.
Fig 4.
Overexpression of RAB11FIP5 inhibits the release of progeny virus.
(A) iSLK.RGB cells were stably transduced with lentiviruses containing a Flag-tagged RAB11FIP5 expression plasmid or an empty vector plasmid and were named iSLK.RGB-RAB11FIP5 or iSLK.RGB-Vector cells, respectively. Overexpression of RAB11FIP5 was detected by western blotting. (B) iSLK.RGB-Vector and iSLK.RGB-RAB11FIP5 cells were treated with dox for different time points as indicated. Extracellular virions were collected from the culture medium and treated with DNase I. Viral DNA was extracted, and KSHV genomic DNA copy numbers were estimated by qPCR by comparison with external standards containing known concentrations of the viral K9 plasmid. (C) Supernatants (500 μl) collected from dox-induced iSLK.RGB-Vector and iSLK.RGB-RAB11FIP5 cells at 72 hpi were incubated with HEK293T cells. The infection rate of HEK293T cells was assessed by fluorescence microscopy. (D) Flow cytometry analysis and quantitation of the percentage of RFP+ cells from (C). (E) Intracellular KSHV genomic DNA was extracted from harvested cells and quantified by qPCR with normalization to GAPDH. (F) RNA was extracted from dox-induced iSLK.RGB-Vector and iSLK.RGB-RAB11FIP5 cells at 72 hpi to measure the transcription level of several KSHV genes: RTA, ORF45, ORF57, ORF65 and LANA. (G) Lysates from dox-induced iSLK.RGB-Vector and iSLK.RGB-RAB11FIP5 cells were analyzed by western blotting at the indicated time points. The expression levels of several KSHV proteins, including ORF45, RTA, ORF64 and ORF65, were determined by immunoblotting with the indicated antibodies.
Fig 5.
Knockdown of endogenous RAB11FIP5 promotes the release of progeny virus.
(A) iSLK.RGB cells were transfected with control siRNA and two RAB11FIP5-specific siRNAs (#1 and #2). The knockdown efficiency was determined by western blotting. (B) iSLK.RGB cells were transfected with control siRNA and siRAB11FIP5-#2. Twenty-four hours after transfection, cells were induced with dox for different time points as indicated. Extracellular virions were collected from the culture medium and treated with DNase I. Viral DNA was extracted, and KSHV genomic DNA copy numbers were estimated by qPCR by comparison with external standards containing known concentrations of the viral K9 plasmid. (C) Supernatants (500 μl) collected from dox-induced cells at 72 hpi were incubated with HEK293T cells. The infection rate of HEK293T cells was examined by fluorescence microscopy. (D) Flow cytometry analysis and quantitation of the percentage of RFP+ cells from (C). (E) Intracellular KSHV genomic DNA was extracted from harvested cells and quantified by qPCR with normalization to GAPDH. (F) The transcription levels of KSHV genes were measured at 72 hpi. (G) The expression levels of several KSHV proteins were determined by immunoblotting at the indicated time post induction.
Fig 6.
RAB11FIP5 promotes lysosomal degradation of ORF45.
(A) Effect of RAB11FIP5 on ORF45 expression. HEK293T cells were cotransfected with 1 μg of ORF45 expression plasmid and increasing amounts of RAB11FIP5 expression vector (0, 0.5, 1, and 2 μg). ORF45 protein expression was assessed by immunoblotting with the indicated antibodies. ORF45 mRNA was detected using RT-qPCR with the indicated primers. (B) Effect of the RAB11FIP5 C2 domain deletion mutant (Δ16–127) on ORF45 expression. HEK293T cells were cotransfected with 1 μg of ORF45 expression plasmid and increasing amounts of the Δ16–127 expression vector (0, 0.5, 1, and 2 μg). ORF45 protein expression was assessed by immunoblotting. ORF45 mRNA was detected using RT-qPCR with the indicated primers. (C) Effects of inhibitors on RAB11FIP5-mediated destabilization of ORF45. HEK293T cells were transfected with the indicated plasmids. Fourteen hours after transfection, cells were treated with the indicated inhibitors for 6 h before immunoblot analysis was performed. (D) HEK293T cells were transiently cotransfected with HA-ORF45 and increasing amounts of Flag-RAB11FIP5 (0, 0.5, 1, and 2 μg). ORF45 protein expression was examined by western blotting in the presence and absence of CHLO. (E) HEK293T cells were transfected with Flag-ORF45 alone or cotransfected with Flag-ORF45 and HA-RAB11FIP5 for 36 h and were then treated with CHLO for another 6 h. A portion of cell samples for whole cell lysis was subjected to western blotting with the indicated antibodies (left panel). The lysosomal fraction was isolated from the remaining cell samples and subjected to western blotting with the indicated antibodies (right panel). (F) HEK293T cells were transfected with ORF45 deletion mutant Flag-Δ115–332 alone or cotransfected with Flag-Δ115–332 and HA-RAB11FIP5 for 36 h and were then treated with CHLO for another 6 h. A portion of cell samples for whole cell lysis was subjected to western blotting with the indicated antibodies (left panel). The lysosomal fraction was isolated from the remaining cell samples and subjected to western blotting with the indicated antibodies (right panel). (G) Stable clones of HeLa-RAB11FIP5 and HeLa-Vector cells were isolated and expanded from the monoclonal cell population by using the limiting dilution method. The expression of RAB11FIP5 was confirmed by western blotting. To assess the effect of RAB11FIP5 on the lysosomal localization of ORF45, stable clones of HeLa-Vector and HeLa-RAB11FIP5 cells were transfected with the ORF45 plasmid. Twenty-four hours after transfection, cells were treated with CHLO for 6 h and were then fixed with 4% paraformaldehyde. Double-label IFA was performed with mouse anti-ORF45 and rabbit anti-LAMP1 antibodies. FITC- and Cy3-conjugated secondary antibodies were used to visualize the labeled LAMP1 and ORF45 proteins, respectively. Images of the colocalization sites were enlarged as shown.
Fig 7.
RAB11FIP5 inhibits ORF45 colocalization with LRs.
(A) HEK293T cells were cotransfected with HA-ORF45 and Flag empty vector for 48 h and were then treated with CHLO for another 6 h. The cells were subjected to the membrane flotation assays as described in the Materials and Methods section. Eleven fractions (lanes 1–11) were collected (top to bottom) from samples subjected to sucrose gradient ultracentrifugation and analyzed by western blotting with specific antibodies as indicated. Caveolin-1 and TfR served as controls that defined the LR (lanes 3–4) and non-LR (lanes 7–11) fractions, respectively. (B) HEK293T cells were cotransfected with HA-ORF45 and Flag-RAB11FIP5 for 48 h and were then treated with CHLO for another 6 h. The cells were subjected to membrane flotation assays as described above. (C) HEK293T cells were cotransfected with HA-ORF45 and RAB11FIP5 C2 domain deletion mutant Flag-Δ16–127 for 48 h and were then treated with CHLO for another 6 h. The cells were subjected to the membrane flotation assays as described above. (D) Stable clones of HeLa-Vector and HeLa-RAB11FIP5 cells were transfected with the ORF45 plasmid. Twenty-four hours after transfection, the cells were treated with CHLO for 6 h and were then incubated with CTB-555 at 37°C for 30 min. Triple-label IFA was performed using CTB-555, rabbit polyclonal anti-GM130 and mouse monoclonal anti-ORF45 antibodies as described in the Materials and Methods section. Alexa Fluor 488-conjugated anti-mouse IgG (green) and Alexa Fluor 647-conjugated anti-rabbit IgG (white) were used as the corresponding secondary antibodies. (E) Stable clones of HeLa-WT and HeLa-RAB11FIP5-/- cells were isolated and expanded from the monoclonal cell population by using the limiting dilution method. RAB11FIP5 knockout was confirmed by western blotting. Stable clones of HeLa-WT and HeLa-RAB11FIP5-/- cells were transfected with the ORF45 plasmid. Twenty-four hours after transfection, the cells were treated with CHLO for 6 h and were then incubated with CTB-555 at 37°C for 30 min. Triple-label IFA was performed as described above.
Fig 8.
RAB11FIP5 impairs the translocation of KSHV particles to the trans-Golgi network.
Stable clones of iSLK-Vector, iSLK-RAB11FIP5, iSLK-WT and iSLK-RAB11FIP5-/- cells were isolated and expended from the monoclonal cell population by the limiting dilution method (A and C). These cells were infected with stock KSHV isolated from BCBL1 cells. Seventy-two hours after infection, latency was established; we designated the corresponding infected cells K/iSLK-Vector, K/iSLK-RAB11FIP5, K/iSLK-WT and K/iSLK-RAB11FIP5-/- cells. (B) K/iSLK-Vector and K/iSLK-RAB11FIP5 cells were induced with dox to stimulate lytic KSHV replication. Viral particles were labeled with the mouse anti-ORF65 antibody, while the trans-Golgi network was labeled with the rabbit anti-TGN46 antibody. FITC- and Cy3-conjugated secondary antibodies were used to visualize the labeled ORF65 and TGN46 proteins, respectively. (D) K/iSLK-WT and K/iSLK-RAB11FIP5-/- cells were induced with dox to stimulate lytic KSHV replication. Viral particles were labeled with the mouse anti-ORF65 antibody, while the trans-Golgi network was labeled with the rabbit anti-TGN46 antibody. FITC- and Cy3-conjugated secondary antibodies were used to visualize the labeled ORF65 and TGN46 proteins, respectively.
Fig 9.
Working model indicating the role of RAB11FIP5 in ORF45-mediated release of KSHV particles.
After budding into the cytoplasm, the tegument protein ORF45 directs tegumented capsids to LRs in the Golgi apparatus and promotes the release of KSHV particles. The host protein RAB11FIP5, a novel ORF45 binding protein, promotes the translocation of ORF45 to lysosomes and increases lysosome-dependent degradation of the ORF45 protein, which impairs the colocalization of ORF45 with LRs in the Golgi apparatus and then impairs the release of KSHV particles through Golgi transport vesicles.
Table 1.
Primers used in this study.