Clathrin is incorporated into MLV and M-PMV virions.
(A) Putative clathrin binding sites proximal to L-domain sequences in MLV p12 and M-PMV pp24 Gag proteins. (B and C) MLV particles incorporate clathrin HC or a coexpressed N-terminal 1–494aa clathrin HC fragment via D156L157L158 motif in the p12 domain. Wild-type or DLL/ALL mutant MLV GagPol expression plasmids were transfected alone (B) or with a plasmid encoding HA tagged clathrin HC 1–494aa (C) in 293T cells. Pelleted virions (B) or cell lysates and virions (C) were subjected to Western blot analysis with anti-MLV CA and anti-clathrin HC (B) or anti-HA antibodies (C). (D and E) M-PMV particles incorporate endogenous clathrin HC or a N-terminal 1–363aa clathrin HC fragment via a D128LISLD133 motif in the pp24 domain. Wild-type or DLISLD/DAASLD mutant M-PMV proviral plasmids (pSARM-4) were transfected alone (D) or with a plasmid encoding an HA tagged clathrin HC 1–363aa (E). Cell lysates and virions were subjected to Western blot analysis with anti-M-PMV CA and anti-clathrin HC (D) or anti-HA (E) antibodies.