Interaction between the G3 and L5 proteins of the vaccinia virus entry-fusion complex

Abstract

The vaccinia virus entry-fusion complex (EFC) consists of 10 to 12 proteins that are embedded in the viral membrane and individually required for fusion with the cell and entry of the core into the cytoplasm. The architecture of the EFC is unknown except for information regarding two pair-wise interactions: A28 with H2 and A16 with G9. Here we used a technique to destabilize the EFC by repressing the expression of individual components and identified a third pair-wise interaction: G3 with L5. These two proteins remained associated under several different EFC destabilization conditions and in each case were immunopurified together as demonstrated by Western blotting. Further evidence for the specific interaction of G3 and L5 was obtained by mass spectrometry. This interaction also occurred when G3 and L5 were expressed in uninfected cells, indicating that no other viral proteins were required. Thus, the present study extends our knowledge of the protein interactions important for EFC assembly and stability.

Fig 1. IP of L5 with G3V5 under EFC destabilization conditions

Human 293TT cells were infected with the following viruses in the presence (+) and absence (−) of IPTG: (A) A16i, (B) A21i, (C) G9i, or (D) vflagJ5i and transfected with vector alone (V) or vector encoding G3V5. The Triton X-100 soluble fraction of infected cells was analyzed by Western blotting before (Input) or after (V5 IP) IP with anti-V5 beads. Antibodies to A16, A21, A28, G3, G9, L5, flag and V5 were used for probing the blot as indicated.

Fig 2. IP of L5 with G3strep under EFC destabilization conditions

(A) Diagram of G3strep recombinant DNA inserted into vA21i. Arrows over the ORFs indicate the direction of transcription. The construct includes the G3L ORF encoding a C-terminal Strep3 tag under the control of the native G3L promoter, a copy of the DsRed ORF under the control of the VACV P11 late promoter, and VACV flanking sequences to allow homologous recombination. (B) Human 293TT cells were infected with vA21i or vA21iG3strep in the presence (+) and absence (−) of IPTG. Triton X-100 soluble extracts were analyzed by Western blotting before (Input) and after strep IP with streptactin beads. Antibodies to A21, A28, G3 and L5 were detected by fluorescence. Antibody to A16 was detected by chemiluminescence.

Fig 3. IP of G3 with L5strep-8xhis under EFC destabilization conditions

(A) Diagram of L5strep-8xhis recombinant DNA inserted into vA21i. Arrows over ORFs indicate the direction of transcription. The construct includes the L5R ORF encoding a C-terminal strep tag followed by 8xhis tag under the control of the native L5R promoter, a copy of the DsRed ORF under the control of the VACV P11 late promoter and VACV flanking sequences to allow homologous recombination. Note: Because of overlap between the downstream ORF of L5 and the upstream ORF of J1 this region (L5/J1) was duplicated in the construct to allow addition of the strep-8xhis tag to the carboxy terminus of L5. Silent mutations were also incorporated in L5/J1 region to prevent instability of the genome due to repeat DNA sequences. (B) Human 293TT cells were infected with vA21i or vA21iL5strep-8xhis (abbreviated vA21iL5strep) in the presence (+) and absence (−) of IPTG. Triton X-100 soluble extracts were analyzed by Western blotting before (Input) and after strep IP with streptactin beads. Antibodies to A21, A28, and L5 were detected by fluorescence. Antibodies to A16 and G3 were detected by chemiluminescence.

Fig 4. Identification by mass spectrometry of EFC proteins associated with G3strep and L5strep-8xhis in the absence of A21

(A) BS-C-1 cells were infected with vA21i or vA21iG3strep with (+) or without (−) IPTG. Infected cells were harvested after 24 h and lysed in 1% triton X-100. After a brief centrifugation the postnuclear supernatant was incubated with streptactin-beads for 3.5 h. Bound protein was eluted with biotin and separated by SDS-PAGE. Protein bands stained with Coomassie blue are shown with the positions of mass marker proteins in kDa on the left. The numbers on the right are the names of proteins that copurified with G3strep as determined by mass spectrometry. Those in parentheses copurified in the absence of A21 expression. Note that multiple proteins comigrated under these conditions and some bands are faint. (B) BS-C-1 cells were infected with vA21i or vA21iL5strep-8xhis (abbreviated vA21iL5strep) with (+) or without (−) IPTG and analyzed as in panel A.

Fig 5. Interaction between G3 and L5 in uninfected cells

(A) Cells were cotransfected with the plasmid encoding L5flag and either the empty vector or the plasmid expressing G3strep. The postnuclear supernatant (Input) and affinity bound proteins were analyzed by SDS-PAGE and Western blotting with antibody to G3 or L5. (B) Cells were cotransfected with the plasmid encoding G3strep and either the empty vector or the plasmid expressing L5flag. The postnuclear supernatant (Input) and affinity bound proteins were analyzed by SDS-PAGE and Western blotting with antibody to G3 or L5.