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1.
Fig 10

Fig 10. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Interactions between the EBV and KSHV NEC proteins. Coinfected Sf21 cells were processed for measurement of Split-Venus fluorescence as described in the legend to Fig. 6. The data derived from two independent infections demonstrate that interactions do occur between the EBV and KSHV NEC proteins.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
2.
Fig 7

Fig 7. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Ultrastructural analysis of infected cells to demonstrate the effects of mutations on nuclear membrane remodeling. Infected Sf21 cells were processed for TEM analysis 68 to 72 h after infection. Bars, 400 nm. The nucleus (n) and cytoplasm (c) are indicated when clearly visible.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
3.
Fig 9

Fig 9. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Expression and cellular localization of the EBV NEC proteins BFRF1 and BFLF2. (A) Western blot analysis of Sf21 cells infected with viruses expressing V5-, GFP-, and mCherry-tagged BFRF1 and BFLF2. Protein standards (kDa) are in lane M. (B) Confocal analysis of the cellular localization of BFRF1 and BFLF2, fused to either GFP or mCherry, in infected Sf21 cells. (C) Colocalization of BFRF1 and BFLF2 at the nuclear margins of coinfected Sf21 cells imaged using confocal microscopy. Magnification for all images, ×100.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
4.
Fig 2

Fig 2. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Biochemical analysis of ORF67 and ORF69 mutant polypeptides. Infected Sf21 cells were harvested at 48 h postinfection, and protein lysates were prepared. Polypeptides were analyzed by NuPage gel electrophoresis (4 to 12% acrylamide), and the membrane following protein transfer was probed with a mouse anti-V5 antibody. Protein standards (kDa) are in lane M.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
5.
Fig 6

Fig 6. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Use of BiFC to quantitate interactions between mutant polypeptides ORF67 and ORF69. Sf21 cells were coinfected with baculoviruses expressing fusions with VenusN and VenusC to quantify both self-interactions (A) and interactions between ORF67 and ORF69 (B). Cells were analyzed for reconstituted Venus fluorescence 48 h postinfection. The data for three independent experiments were plotted. In panel A, the two viruses encoded either VenusN or VenusC in the same wild-type or mutant background, whereas in panel B, the ORF67-expressing viruses used were VenusC fusions and the ORF69-expressing viruses were VenusN fusions. 67VN and 69VN are single infections.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
6.
Fig 4

Fig 4. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Localization and interactions of the polypeptide truncation mutants of ORF67. Infections similar to those described in the Fig. 3 legend were carried out using the ORF67 T240 and T264 mutants. (A) The distribution and interactions of ORF67 T240 fused to GFP were examined by confocal microscopy following infection for 48 h. (B) Colocalization of ORF67 T264mCherry and ORF69GFP at the nuclear margins. Magnification, ×100. For panel B and lower images of panel A, the scans from both the green and red channels are shown as well as the digitally merged images.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
7.
Fig 5

Fig 5. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Split-Venus fluorescence reconstitution by interactions of the KSHV NEC. Sf21 cells were infected with viruses expressing fusions with VenusN and VenusC. (A and B) A significant amplification of fluorescence was observed and visualized by confocal microscopy for both self-interactions (A) and interactions between ORF67 and ORF69 (B). The Venus fluorescence observed was similar to the distribution of GFP-tagged ORF67 and ORF69 (magnification, ×63). (C) These interactions were quantitated by measuring fluorescence as a consequence of Venus reconstitution following coinfection of Sf21 cells with viruses expressing VenusN and VenusC fusions. Fluorescence produced was measured after 48 h of infection, and the measurements of three independent infections were plotted.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
8.
Fig 3

Fig 3. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Colocalization of ORF67 and ORF69 mutants at the nuclear margins. Sf21 cells were coinfected with baculoviruses expressing GFP and mCherry fusion proteins to discover whether the mutant proteins colocalize with the wild-type partner of the NEC by confocal microscopy. (A) Wild-type (WT) and mutant ORF67 polypeptides fused to GFP were examined in the presence of wild-type ORF69mCherry 48 h following infection. Magnification, ×63. (B) The distribution of wild-type and mutant ORF69 polypeptides fused to GFP was examined in the presence of wild-type ORF67mCherry, in this case 68 h after infection. Magnification, ×100. The scans from both the green and red channels are shown as well as the digitally merged images.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
9.
Fig 11

Fig 11. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Remodeling of cellular membranes by the EBV and KSHV NEC. Sf21 cells were infected with the baculovirus expressing the EBV BFRF1 or coinfected with baculoviruses expressing the EBV and KSHV NEC proteins. Infected cells were processed for electron microscopy 72 h after infection. ORF69 was able to remodel nuclear membrane proliferations induced by either BFRF1 or ORF67; however, EBV BFLF2 did not display the same activity. Bars, all 400 nm, except for that in the ORF67+ORF69 panel, which is 200 nm. The nucleus (n) and cytoplasm (c) are indicated. The bottom right panel shows the results of Western blot analysis of Sf21 cells infected with combinations of the KSHV and EBV NEC-expressing viruses. The blot was probed with anti-V5 antibody. Protein standards (kDa) are shown in lane M.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
10.
Fig 1

Fig 1. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

Polypeptide sequences of ORF67 and ORF69 and mutations made in these proteins. Shown are the amino acid sequences of KSHV ORF67 and ORF69 (strain BCBL-1 BAC36) (59). The in-frame deletions made are highlighted by black boxes, and the positions of the polypeptide truncation mutations are indicated by the arrows (T240 and T264; truncations after amino acids 239 and 263, respectively). The transmembrane domain residues of ORF67 predicted using MacVector are shown in bold. Underlined residues are those that are conserved in the gammaherpesviruses (KSHV, EBV, rhesus rhadinovirus, murine herpesvirus 68, and herpesvirus saimiri). Residues that are completely conserved in all human herpesvirus proteins that are located close to or within the in-frame deletions are in red. The amphipathic alpha-helix in ORF67 predicted by Milbradt et al. (29) is marked by a purple box, as is an alpha-helix in the N terminus of ORF69 that was determined using MacVector software. The arginine-rich region in the N terminus of ORF69, which may contain a nuclear localization signal (NLS), is shown in a green box.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.
11.
Fig 8

Fig 8. From: Interactions of the Kaposi's Sarcoma-Associated Herpesvirus Nuclear Egress Complex: ORF69 Is a Potent Factor for Remodeling Cellular Membranes.

The KSHV NEC proteins colocalize at nuclear margins of mammalian cells and alter these membranes. (A) HEK-293T cells were transfected with plasmids expressing ORF67GFP or ORF69mCherry. (B) Cells were cotransfected with a plasmid expressing ORF67GFP and a plasmid expressing ORF69mCherry. The cells were imaged by confocal microscopy at 24 h posttransfection. (C) Similar cultures cotransfected with ORF67GFP- and ORF69V5-expressing plasmids demonstrate cellular membrane remodeling events. (D) HEK-293T cells were transfected with ORF69GFP-expressing plasmids and imaged by confocal microscopy 24 h posttransfection. A potential nuclear localization sequence within ORF69 is influenced by changes in arginines at positions R17, R21, R24, and R25. (E) Sequence of the N-terminal 50 amino acids of ORF69 showing the arginines that were sequentially changed to alanine. For example, R21-39 has alanine substitutions at all arginines between residues 21 and 39. Magnifications, ×63 (A and D) and ×100 (B). Bars in panel C are 200 nm, except for that in the center image, which is 100 nm. The nuclear (n) and cytoplasmic (c) compartments are indicated.

Eric M. Luitweiler, et al. J Virol. 2013 April;87(7):3915-3929.

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