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. 2011 Jan 19;85(7):3631–3641. doi: 10.1128/JVI.01984-10

FIG. 2.

FIG. 2.

The Mopeia virus Z protein YLCL motif is required for NP incorporation into Z-induced VLPs. (A) Alignment of the following arenavirus Z protein sequences showing the conserved YLCL region (the corresponding NCBI accession numbers are shown in parentheses): wild-type Mopeia virus (WT MOPV) Z (accession number AAV54106), mutant Mopeia virus (Mut MOPV) Z containing alanine substitutions at amino acids 53 to 56, Lassa virus (LASV) Z (accession number NP_694871), lymphocytic choriomeningitis virus (LCMV) Z (accession number P18541), Lujo virus (LUJV) Z (accession number YP_002929492), Junin virus (JUNV) Z (accession number Q6IVU5), Machupo virus (MACV) Z (accession number AAY27823), Guanarito virus (GTOV) Z (accession number NP_899220), Chapare virus (CV) Z (accession number YP_001816784), and Tacaribe virus (TCRV) Z (accession number Q88470). The numbers indicate the numbers of amino acids within the respective Z proteins. (B) Comparison of the stability between wild-type Z and Z-AAAA. We replaced the glycine residues at position 2 with alanines, generating Z-G2A and Z-AAAA/G2A, respectively. The constructs were individually transfected into 293T cells. After 16, 32, and 48 h, cellular lysates were examined for Z protein expression by use of Western blotting with an anti-HA antibody. An equal number of cells in the samples was confirmed by using Western blotting with an anti-actin antibody. (C) pC-MopZ-HA or pC-MopZ-AAAA-HA was individually transfected into 293T cells. VLP isolation and analysis by use of Western blotting with an anti-HA antibody were performed 48 h after transfection. The VLP formation efficiency of Z was calculated as the amount of Z in the VLPs divided by that in the lysates. The budding efficiency for wild-type Z was set at 1, and the efficiency for mutant Z is reported relative to the wild-type result. Three independent experiments were performed, and standard deviations were calculated. Representative data are shown. (D) 293T cells were cotransfected with plasmids encoding pC-MopNP-FLAG and either the wild type or Z-AAAA. After 48 h, VLPs were isolated and analyzed by Western blotting with anti-FLAG and anti-HA antibodies. The VLP formation efficiency of Z was calculated as described above (C). The NP incorporation efficiency was calculated as the ratio of the amount of the NP protein relative to that of the Z protein detected in the VLPs. The averages of data from three independent experiments are shown. (E) pC-MopZ-HA, pC-MopZ-AAAA-HA, pC-MopZ-ALCL-HA, pC-MopZ-YACL-HA, or pC-MopZ-YLCA-HA was cotransfected with pC-MopNP-FLAG into 293T cells. VLP isolation and analysis were performed as described above (D).